Human monoclonal antibodies to herpes simplex virus and methods therefor

ABSTRACT

The present invention describes human monoclonal antibodies which immunoreact with Herpes simplex virus Type-1 and Type-2. Also disclosed are immunotherapeutic and diagnostic methods of using the monoclonal antibodies, as well as nucleic acids and cell lines for producing the monoclonal antibodies.

This invention was made with Government support under grant No. 1RO1A133292-01 ARRA and NIMH 47680 from the National Institutes of Health.The Government has certain rights in this invention.

This is a continuation of application Ser. No. 08/178,201, filed Jan. 4,1994, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to field of immunology and specificallyto human monoclonal antibodies which bind and neutralize Herpes simplexvirus (HSV) Type-1 and Type-2.

2. Description of Related Art

Herpes simplex virus (HSV) remains one of the most common viral maladiesin man, achieving a worldwide distribution and causing a variety ofinfections. Two forms of the virus, HSV Type-1 (HSV-1) and HSV Type-2(HSV-2), have been distinguished by clinical, biochemical, andserological criteria. HSV-2 is more commonly implicated in genitalinfection, while HSV-1 is associated with oral and ocular disease. Bothtypes of the virus may become latent after traveling intranasally tosensory ganglia.

Primary and secondary herpes infections in individuals immunocompromisedby underlying disease or immunosupressive drugs are often more severethan in the normal host. Such individuals, which include AIDS patients,those with hematological or lymphoreticular neoplasms, (Siegal, et al.,N. Engl. J. Med., 305:1439-1444, 1981; Greenberg, et al., J. Infect.Dis., 156:280-287, 1987; Buss, et al., J.A.M.A., 243:1903-1905, 1980)and organ and bone marrow transplant recipients, are also prone toincreased frequency of secondary herpes episodes. In the case oftransplant recipients, the severity of herpes infection correlates withthe degree of immunosuppressive therapy employed (Rand, et al., N. Engl.J. Med., 296:1372-1377, 1977).

Devastating illness may also result from HSV infection of the neonate.In the U.S., such infections are encountered in 1 in 2,500 to 1 in 5,000deliveries per year, most being acquired following intrapartum contactwith infected genital secretions (Whitley, Virology eds., 2ndEds:1843-1889, 1990). As compared to a recurrent episode, primaryinfection occurring late in pregnancy generally produces more frequentand severe disease in the newborn. This correlates with a greatermaternal viral load at delivery (Corey, et al., Ann. Intem. Med.,98:958-972, 1983), probably arising because the immune reponse to thevirus is only in its early stages.

Current therapy of herpes infections is limited, although acyclovir,vidarabine and related drugs have proven useful for the management ofspecific infections such as mucocutaneous herpes infections in theimmunocompromised host, herpes simplex encephalitis and neonatal herpes.Recurrent episodes, however, are less responsive. Moreover, viralstrains resistant to these drugs have been isolated fromimmunocompromised patients (Englund, et al., Ann. Int. Med.,112:416-422, 1990; Sacks, et al., supra; Erlich, et al., N. Engl. J.Med., 320:293-296, 1989). A potential alternative approcah to HSVprevention and therapy is offered by specific human antibodies to thevirus. Such reagents, if highly efficient in virus neutralization and inmediating the clearance of virally infected cells, may prove useful inreplacing or complementing existing clinical regimes.

There is evidence of a significant protective role for antibody in humaninfection in vivo. The presence of neutralizing antibody in acute phaseserum during primary infections has been associated with reducedseverity and duration of the primarly genital herpes episode (Core,J.A.M.A., 248:1041-1049, 1982). Further, it has been shown that thedevelopment of recurrent genital herpes in an individual following aprimary infection with homologous virus is inversely correlated with thetiter of HSV-2 neutralizing antibody in the convalescent serum (Reeves,et al., N. Engl. J. Med., 305:315-319, 1981). Moreover, the titer ofanti-HSV antibodies in bone marrow transplant recipients is predictiveof the risk of infection (Pass, et al., J. Infect. Dis., 140:487-491,1979). In vitro, human serum antibody has additionally been shown toneutralize extra-cellular virus and lyse certain HSV-infected cells(Allison, Transplant Ref., 19:3-55, 1974).

HSV is a complex virus. Over 50 virus encoded polypeptides, includingboth structural (envelope and core) and regulatory proteins, have beenidentified in infected cells. Analysis of the humoral response againstHSV and the identification and characterization of potentiallyprotective antigens has been undertaken largely using human sera andmouse monoclonal antibodies (Fujinaga, et al., J. Infect. Dis.,155:45-53, 1987; Kapoor, et al., J. Gen. Virol., 60:225-233, 1982;Kumel, et al., J. Virol., 56:930-937, 1985; Rector, et al., J. Fen.Virol., 65:657-661, 1984; Simmons, et al., J. Virol., 53:944-948, 1985;Kuhn, et al., J. Med. Virol., 23:135-150, 1987). The main targets of thehumoral and cellular responses appear to be the 7 well characterized HSVenvelope glycoproteins, gB, gC, gD, gE, gG, gH and gl, which are foundboth on the virion and on the infected cell surface where they arethought to promote viral attachment and penetration through multipleinteractions between themselves and the cell membrane. Only gB, gD andgH have been found to be indispensable for viral growth in cell culture.Virus mutants defective in these molecules will bind to the host cellsurface, but are unable to penetrate into the cytoplasm (Desai, et al.,J. Gen. Virol., 69:1147-1156, 1988; Fuller, et al., J. Virol.,63:3435-3443, 1989; Ugas, et al., J. Virol., 62:1486-1494, 1988; Weber,et al., Science, 236:576579, 1987). Those glycoproteins which aremandatory for virus attachment have not yet been identified.

Each envelope glycoprotein is capable of eliciting mouse monoclonalantibodies able to neutralize virus in vitro (Whitley, et al., Virologyeds., 2nd Ed:1843-1889, 1990). Passive immunization with monoclonalantibodies specific for gB, gC, gD, gE and gH has also been shown toprotect animals from infection (Kumel, et al., J. Virol., 56:930-937,1985; Simmons, et al., J. Virol., 53:944-948, 1985; Balachandran, etal., Infect. Immun., 37:1132-1137, 1982; Dix, et al., Infect Immun.,34:192-199, 1981). In addition, polyclonal immune sera and gD specificmonoclonal antibodies have been shown to protect mice from recurrentdisease (Simmons, et al., J. Virol., 53:944-948, 1985). It has also beendemonstrated that monoclonal antibodies against gB and gE suppress thereplication of HSV-1 in trigeminal ganglia (Oakes, et al., J. Virol.,51:656-661, 1984). Glycoproteins B and D appear to elicit a major partof the antibody response to virus in humans and also appear to be themajor targets of neutralizing antibodies (Kuhn, et al., J. Med. Virol.,23:135-150, 1987).

There is thus a considerable body of evidence illustrating the potentialvalue of monoclonal antibodies as agents for immune prophylaxis andtherapy in HSV infection. The development of combinatorial antibodylibraries displayed on the surface of phage offers the possibility ofaccessing monoclonal human antibody specificities against infectiousagents from any individual with clearly demonstrable serum antibodiesagainst the pathogen (Williamson, et al., Proc. Nat'l Acad. Sci. USA,90:4141-4145, 1993). In the present invention, a library from a longterm asymptomatic HIV-1 positive individual with serum antibody titeragainst HSV-1 and HSV-2 was used to isolate a diverse array of humanmonoclonal antibodies specific for these two viruses. The generation ofpanels of antibodies using this methodology permits a thoroughcharacterization of the human antibody response to these pathogens andfurther identify particular antibodies of potential value for clinicalapplications. The present invention provides such an HSV neutralizinghuman antibody.

SUMMARY OF THE INVENTION

The present invention provides human monoclonal antibodies which bindand neutralize Herpes simplex virus (HSV) Type-1 and Type-2 and celllines which produce these monoclonal antibodies. The antibodies of theinvention were identified using phagemid vectors which allowidentification and isolation of human monoclonal antibodies thatimmunoreact with HSV from combinatorial libraries. These phagemidvectors allow the rapid preparation of large numbers of neutralizingantibodies. The identified neutralizing antibodies may define newepitopes on HSV, thereby increasing the availability of newimmunotherapeutic human monoclonal antibodies to HSV-associateddiseases.

Also provided are amino acid sequences which confer HSV-1 and HSV-2neutralization function to the paratope of these monoclonal antibodiesand which can be used immunogenically to identify other antibodies thatspecifically bind and neutralize HSV. The monoclonal antibodies of theinvention find particular utility as reagents for the diagnosis andimmunotherapy of HSV disease.

A major advantage of the monoclonal antibodies of the invention derivesfrom the fact that they are encoded by a human polynucleotide sequence.Thus, in vivo use of the monoclonal antibodies of the invention fordiagnosis and immunotherapy of HSV disease greatly reduces the problemsof significant host immune response to the passively administeredantibodies which is a problem commonly encountered when monoclonalantibodies of xenogeneic or chimeric derivation are utilized.

The antibodies of the invention have extremely high neutralizingactivity and may be particularly efficacious in ameliorating HSV diseasewhen administered topically for genital, oral and ocular herpes. Theability to utilize Fab fragments in vivo for systemic protection fromHSV infections provides significant advantages over the use of wholeantibody molecules such as: (1) greater tissue penetration; (2)avoidance of effector functions associated with Fc, such asinflammation; and (3) rapid clearance.

In one embodiment, the invention contemplates a human monoclonalantibody capable of immunoreacting with Herpes simplex virus (HSV), andpreferably neutralizes HSV. A preferred human monoclonal antibody hasthe binding specificity of a monoclonal antibody comprising a heavychain immunoglobulin variable region amino acid residue sequence asfound in SEQUENCE ID No. 1, and conservative substitutions thereof.

In another embodiment, the invention describes a polynucleotide sequenceencoding a heavy or light chain immunoglobulin variable region aminoacid residue sequence portion of a human monoclonal antibody of thisinvention. Also contemplated are DNA expression vectors containing thepolynucleotide, and host cells containing the vectors andpolynucleotides of the invention.

The invention also contemplates a method of detecting Herpes simplexvirus (HSV) comprising contacting a sample suspected of containing HSVwith a diagnostically effective amount of the monoclonal antibody ofthis invention, and determining whether the monoclonal antibodyimmunoreacts with the sample. The method can be practiced in vitro or invivo, and may include a variety of methods for determining the presenceof an immunoreaction product.

In another embodiment, the invention describes a method for providingpassive immunotherapy to Herpes simplex virus (HSV) disease in a human,comprising administering to the human an immunotherapeutically effectiveamount of the monoclonal antibody of this invention. The administrationcan be provided prophylactically, and by a parenteral administration.Pharmaceutical compositions containing one or more of the differenthuman monoclonal antibodies are described for use in the therapeuticmethods of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the neutralizing activity of Fab8, as measured by plaquereduction. FIG. 1A shows activity against HSV-1 and FIG. 1B showsactivity against HSV-2. Purified Fab8 neutralized HSV-1 with a 50%inhibition at about 0.25 μg/ml and with an 80% inhibition at 0.6 μg/ml,while HSV-2 was neutralized with a 50% inhibition at about 0.05 μg/mland an 80% inhibition at 0.1 μg/ml.

FIG. 2 shows an inhibition of plaque development assay. Purified Fab8inhibited the development of plaques when applied 4 hours post-infection(hpi) on monolayers infected with HSV-1 (FIG. 2A, FIG. 2B) or HSV-2(FIG. 2C, FIG. 2D) 4 hours post infection. FIG. 2A shows statisticallysignificant reduction in plaque size was observed at concentrations of 5and 1 μg/ml (*=p<0.01), with an approximate 50% reduction in plaque sizeat 5 μg/ml. The number of plaques was also dramatically reduced at Fabconcentrations of 5 and 25 μg/ml (FIG. 2B, FIG. 2D). At 25 μg/ml and 72hrs hpi plaque development in HSV-2 infected monolayers was completelyinhibited (FIG. 2C, FIG. 2D). FIG. 2E shows an inhibition of plaquedevelopment assay with HSV-2 infected monolayers at a number ofdifferent Fab concentrations 86 hpi.

FIG. 3 shows a post-attachment neutralization assay. Fab8 reduced HSV-1infectivity after virion attachment. FIG. 3A shows the percentage ofplaque reduction pre- and post-attachment at different Fabconcentrations. FIG. 3B shows the post-/pre-attachment neutralizationratio at different Fab concentrations.

FIG. 4 shows the identification of the protein recognized by Fab8.SDS-PAGE of total proteins from HSV-2 infected Vero cells (lanes 1) andof the product of immunoprecipitation with Fab8 (lanes 2). Western blotsperformed in parallel were probed with a mouse monoclonal anti gDantibody (MAB α-gD) and for the purpose of control, a rabbit polyclonalanti-HSV-2 preparation (RAB α-HSV2). The Coomassie stain of a gel run inparallel is also shown. Fab8 immunoprecipitated a band of apparentmolecular weight 48-50 kD which was recognized by a mouse monoclonalspecific for gD, but not by mouse monoclonal antibodies against otherHSV glycoproteins.

FIG. 5A shows the kinetics of neutralization of HSV Type-2, strain G(ATCC, Rockville, Md.) with Fab8. V/Vo is the number of viral plaqueforming units present at time 0 (Vo) or after neutralization (V).

FIG. 5B shows the kinetics of neutralization of HSV Type-2, strain G(ATCC, Rockville, Md.) with Fab8. The neutralization rate is plottedversus the antibody concentration indicating first order kinetics.

FIG. 6 shows the kinetics of neutralization of HSV Type-2, strain G(ATCC, Rockville, Md.) with Fab8. The plaque reduction is plotted versusantibody concentration for 6 different clinical isolates.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to human monoclonal antibodies which arespecific for, and neutralize Herpes simplex virus (HSV) Type-1 andType-2. In a preferred embodiment of the invention, human monoclonalantibodies are disclosed which are capable of binding epitopicpolypeptide sequences in glycoprotein D of HSV. Also disclosed is anantibody heavy chain amino acid sequence which is highly specific andconfers neutralization of HSV when the virus is bound by antibodieshaving the amino acid sequence. This specificity enables the humanmonoclonal antibody, and human monoclonal antibodies with likespecificity, to be used in the diagnosis and immunotherapy of HSVdisease.

The term "HSV disease" means any disease caused, directly or indirectly,by HSV as well as diseases which predispose a patient to infection byHSV. Examples of diseases falling into the former category includegenital, oral and ocular herpes. Diseases in the latter category (i.e.,those which place the patient at risk of severe HSV infection) include,generally, any condition that causes a state of immunosuppression ordecreased function of the immune system such as patients who receiveorgan transplants, AIDS patients, those with hematological orlymphoreticular neoplasms, and infants.

In one aspect, the present invention is directed to combinatoriallyderived human monoclonal antibodies which are reactive with and bind anHSV neutralization site and cell lines which produce such antibodies.The isolation of cell lines producing monoclonal antibodies of theinvention can be accomplished using routine screening techniques whichpermit determination of the elementary reaction pattern of themonoclonal antibody of interest. Thus, if a human monoclonal antibodybeing tested binds and neutralizes members of Type-1 and Type-2 HSV inthe same manner as the human monoclonal antibodies of the invention,then the human monoclonal antibody being tested and the human monoclonalantibody produced by the cell lines of the invention are equivalent.

It is also possible to determine, without undue experimentation, if ahuman monoclonal antibody has the same specificity as a human monoclonalantibody of the invention by ascertaining whether the former preventsthe latter from binding to HSV. If the human monoclonal antibody beingtested competes with the human monoclonal antibody of the invention, asshown by a decrease in binding by the human monoclonal antibody of theinvention, then it is likely that the two monoclonal antibodies bind tothe same, or a closely related, epitope.

Still another way to determine whether a human monoclonal antibody hasthe specificity of a human monoclonal antibody of the invention is topre-incubate the human monoclonal antibody of the invention with HSVwith which it is normally reactive, and then add the human monoclonalantibody being tested to determine if the human monoclonal antibodybeing tested is inhibited in its ability to bind HSV. If the humanmonoclonal antibody being tested is inhibited then, in all likelihood,it has the same, or functionally equivalent, epitopic specificity as themonoclonal antibody of the invention. Screening of human monoclonalantibodies of the invention, can be also carried out utilizing HSV anddetermining whether the monoclonal antibody neutralizes HSV.

By using the human monoclonal antibodies of the invention, it is nowpossible to produce anti-idiotypic antibodies which can be used toscreen human monoclonal antibodies to identify whether the antibody hasthe same binding specificity as a human monoclonal antibody of theinvention and also used for active immunization (Herlyn, et al.,Science, 232:100, 1986). Such anti-idiotypic antibodies can be producedusing well-known hybridoma techniques (Kohler and Milstein, Nature,256:495, 1975). An anti-idiotypic antibody is an antibody whichrecognizes unique determinants present on the human monoclonal antibodyproduced by the cell line of interest. These determinants are located inthe hypervariable region of the antibody. It is this region which bindsto a given epitope and, thus, is responsible for the specificity of theantibody. An anti-idiotypic antibody can be prepared by immunizing ananimal with the monoclonal antibody of interest. The immunized animalwill recognize and respond to the idiotypic determinants of theimmunizing antibody and produce an antibody to these idiotypicdeterminants. By using the anti-idiotypic antibodies of the immunizedanimal, which are specific for the human monoclonal antibody of theinvention produced by a cell line which was used to immunize the secondanimal, it is now possible to identify other clones with the sameidiotype as the antibody of the hybridoma used for immunization.Idiotypic identity between human monoclonal antibodies of two cell linesdemonstrates that the two monoclonal antibodies are the same withrespect to their recognition of the same epitopic determinant. Thus, byusing anti-idiotypic antibodies, it is possible to identify otherhybridomas expressing monoclonal antibodies having the same epitopicspecificity.

It is also possible to use the anti-idiotype technology to producemonoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is the"image" of the epitope bound by the first monoclonal antibody. Thus, theanti-idiotypic monoclonal antibody can be used for immunization, sincethe anti-idiotype monoclonal antibody binding domain effectively acts asan antigen.

The term "antibody" as used in this invention includes intact moleculesas well as fragments thereof, such as Fab and F(ab')₂, which are capableof binding the epitopic determinant. In the present invention Fabfragments are preferred. Fabs offer several advantages over F(ab')₂ sand whole immunoglobulin molecules as a therapeutic modality. First,because Fabs have only one binding site for their cognate antigen, theformation of immune complexes is precluded, whereas such complexes canbe generated when divalent F(ab')₂ s and whole immunoglobulin moleculesencounter their target antigen. This is of some importance becauseimmune complex deposition in tissues can produce adverse inflammatoryreactions. Second, since Fabs lack an Fc region they cannot triggeradverse inflammatory reactions that are activated by Fc, such asinitiation of the complement cascade. Third, the tissue penetration ofthe small Fab molecule is likely to be much better than that of thelarger whole antibody. Fourth, Fabs can be produced easily andinexpensively in bacteria, such as E. coli, whereas whole immunoglobulinantibody molecules require mammalian cells for their production inuseful amounts. The latter entails transfection of immunoglobulinsequences into mammalian cells with resultant transformation.Amplification of these sequences must then be achieved by rigorousselective procedures and stable transformants must be identified andmaintained. The whole immunoglobulin molecules must be produced bystably transformed, high expression cells in culture with the attendantproblems of serum-containing culture medium. In contrast, production ofFabs in E. coli eliminates these difficulties and makes it possible toproduce these antibody fragments in large fermenters which are lessexpensive than cell culture-derived products.

In addition to Fabs, smaller antibody fragments and epitope-bindingpeptides having binding specificity for at least one epitope of HSV,preferably on glycoprotein D of HSV, are also contemplated by thepresent invention and can also be used to neutralize the virus. Forexample, single chain antibodies can be constructed according to themethod of U.S. Pat. No. 4,946,778 to Ladner et al., which isincorporated herein by reference in its entirety. Single chainantibodies comprise the variable regions of the light and heavy chainsjoined by a flexible linker moiety. Yet smaller is the antibody fragmentknown as the single domain antibody, which comprises an isolate VHsingle domain. Techniques for obtaining a single domain antibody with atleast some of the binding specificity of the intact antibody from whichthey are derived are known in the art. For instance, Ward, et al., in"Binding Activities of a Repertoire of Single Immunoglobulin VariableDomains Secreted from Escheria coli," Nature 341:644-646, disclose amethod for screening to obtain an antibody heavy chain variable region(VH single domain antibody) with sufficient affinity for its targetepitope to bind thereto in isolate form.

The monoclonal antibodies of the invention are suited for in vitro use,for example, in immunoassays in which they can be utilized in liquidphase or bound to a solid phase carrier. In addition, the monoclonalantibodies in these immunoassays can be detectably labeled in variousways. Examples of types of immunoassays which can utilize monoclonalantibodies of the invention are competitive and non-competitiveimmunoassays in either a direct or indirect format. Examples of suchimmunoassays are the radioimmunoassay (RIA) and the sandwich(immunometric) assay. Detection of the antigens using the monoclonalantibodies of the invention can be done utilizing immunoassays which arerun in either the forward, reverse, or simultaneous modes, includingimmunohistochemical assays on physiological samples. Those of skill inthe art will know, or can readily discern, other immunoassay formatswithout undue experimentation.

The monoclonal antibodies of the invention can be bound to manydifferent carriers and used to detect the presence of HSV. Examples ofwell-known carriers include glass, polystyrene, polypropylene,polyethylene, dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, agaroses and magnetite. The nature of the carrier canbe either soluble or insoluble for purposes of the invention. Thoseskilled in the art will know of other suitable carriers for bindingmonoclonal antibodies, or will be able to ascertain such, using routineexperimentation.

There are many different labels and methods of labeling known to thoseof ordinary skill in the art. Examples of the types of labels which canbe used in the present invention include enzymes, radioisotopes,fluorescent compounds, colloidal metals, chemiluminescent compounds, andbio-luminescent compounds. Those of ordinary skill in the art will knowof other suitable labels for binding to the monoclonal antibodies of theinvention, or will be able to ascertain such, using routineexperimentation. Furthermore, the binding of these labels to themonoclonal antibodies of the invention can be done using standardtechniques common to those of ordinary skill in the art.

For purposes of the invention, HSV may be detected by the monoclonalantibodies of the invention when present in biological fluids andtissues. Any sample containing a detectable amount of HSV can be used. Asample can be a liquid such as urine, saliva, cerebrospinal fluid,blood, serum and the like, or a solid or semi-solid such as tissues, or,alternatively, a solid tissue such as those commonly used inhistological diagnosis. For detection of HSV, preferred samples includea swab from a mucocutaneous lesion, a needle aspiration from a freshvesicle, cerebrospinal fluid, brain tissue homogenate (postmortem) andurine in exceptional cases where genital herpes is complicated byurethritis or cystitis.

Another labeling technique which may result in greater sensitivityconsists of coupling the antibodies to low molecular weight haptens.These haptens can then be specifically detected by means of a secondreaction. For example, it is common to use haptens such as biotin, whichreacts with avidin, or dinitrophenol, pyridoxal, or fluorescein, whichcan react with specific anti-hapten antibodies.

As used in this invention, the term "epitope" means any antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics.

The materials for use in the assay of the invention are ideally suitedfor the preparation of a kit. Such a kit may comprise a carrier meansbeing compartmentalized to receive in close confinement one or morecontainer means such as vials, tubes, and the like, each of thecontainer means comprising one of the separate elements to be used inthe method. For example, one of the container means may comprise a humanmonoclonal antibody of the invention which is, or can be, detectablylabelled. The kit may also have containers containing buffer(s) and/or acontainer comprising a reporter-means, such as a biotin-binding protein,such as avidin or streptavidin, bound to a reporter molecule, such as anenzymatic, or fluorescent label.

In using the human monoclonal antibodies of the invention for the invivo detection of antigen, the detectably labeled monoclonal antibody isgiven in a dose which is diagnostically effective. The term"diagnostically effective" means that the amount of detectably labeledhuman monoclonal antibody is administered in sufficient quantity toenable detection of the site having the HSV antigen for which themonoclonal antibodies are specific.

The concentration of detectably labeled human monoclonal antibody whichis administered should be sufficient such that the binding to HSV isdetectable compared to the background. Further, it is desirable that thedetectably labeled monoclonal antibody be rapidly cleared from thecirculatory system in order to give the best target-to-background signalratio.

As a rule, the dosage of detectably labeled human monoclonal antibodyfor in vivo diagnosis will vary depending on such factors as age, sex,and extent of disease of the individual. The dosage of human monoclonalantibody can vary from about 0.01 mg/m² to about 500 mg/m², preferably0.1 mg/m² to about 200 mg/m², most preferably about 0.1 mg/m² to about10 mg/m². Such dosages may vary, for example, depending on whethermultiple injections are given, tissue, and other factors known to thoseof skill in the art.

For in vivo diagnostic imaging, the type of detection instrumentavailable is a major factor in selecting a given radioisotope. Theradioisotope chosen must have a type of decay which is detectable for agiven type of instrument. Still another important factor in selecting aradioisotope for in vivo diagnosis is that the half-life of theradioisotope be long enough so that it is still detectable at the timeof maximum uptake by the target, but short enough so that deleteriousradiation with respect to the host is minimized. Ideally, a radioisotopeused for in vivo imaging will lack a particle emission, but produce alarge number of photons in the 140-250 keV range, which may be readilydetected by conventional gamma cameras.

For in vivo diagnosis radioisotopes may be bound to immunoglobulineither directly or indirectly by using an intermediate functional group.Intermediate functional groups which often are used to bindradioisotopes which exist as metallic ions to immunoglobulins are thebifunctional chelating agents such as diethylenetriaminepentacetic acid(DTPA) and ethylenediaminetetraacetic acid (EDTA) and similar molecules.Typical examples of metallic ions which can be bound to the monoclonalantibodies of the invention are ¹¹¹ In, ⁹⁷ Ru, ⁶⁷ Ga, ⁶⁸ Ga, ⁷² As, ⁸⁹Zr, and ²⁰¹ TI.

The monoclonal antibodies of the invention can also be labeled with aparamagnetic isotope for purposes of in vivo diagnosis, as in magneticresonance imaging (MRI) or electron spin resonance (ESR). In general,any conventional method for visualizing diagnostic imaging can beutilized. Usually gamma and positron emitting radioisotopes are used forcamera imaging and paramagnetic isotopes for MRI. Elements which areparticularly useful in such techniques include ¹⁵⁷ Gd, ⁵⁵ Mn, 162Dy, ⁵²Cr, and ⁵⁶ Fe.

The human monoclonal antibodies of the invention can be used in vitroand in vivo to monitor the course of HSV disease therapy. Thus, forexample, by measuring the increase or decrease in the number of cellsinfected with HSV or changes in the concentration of HSV present in thebody or in various body fluids, it would be possible to determinewhether a particular therapeutic regimen aimed at ameliorating the HSVdisease is effective.

The human monoclonal antibodies can also be used immunotherapeuticallyfor HSV disease. The term "immunotherapeutically" or "immunotherapy" asused herein in conjunction with the monoclonal antibodies of theinvention denotes both prophylactic as well as therapeuticadministration. Thus, the monoclonal antibodies can be administered tohigh-risk patients such as AIDS patients, in order to lessen thelikelihood and/or severity of HSV disease, or administered to patientsalready evidencing active HSV infection.

The dosage ranges for the immunotherapeutic administration of themonoclonal antibodies of the invention are those large enough to producethe desired effect in which the symptoms of the HSV disease areameliorated or the likelihood of infection is decreased. The dosageshould not be so large as to cause adverse side effects, such ashyperviscosity syndromes, pulmonary edema, conjestive heart failure, andthe like. Generally, the dosage will vary with the age, condition, sexand extent of the disease in the patient and can be determined by one ofskill in the art. The dosage can be adjusted by the individual physicianin the event of any complication. Dosage can vary from about 0.01 mg/kgto about 300 mg/kg, preferably from about 0.1 mg/kg to about 200 mg/kg,most preferably from about 0.2 mg/kg to about 20 mg/kg, in one or moredose administrations daily, for one or several days. Preferred isadministration of the antibody for 2 to 5 or more consecutive days inorder to avoid "rebound" of virus replication from occurring.

The human monoclonal antibodies of the invention can be administeredparenterally by injection or by gradual infusion over time. The humanmonoclonal antibodies of the invention can be administeredintravenously, intraperitoneally, intramuscularly, subcutaneously, orintracavity. A formulation containing the monoclonal antibodies of theinvention may also be administered externally to a subject, at the siteof the disease for exertion of local or transdermal action. When usedtherapeutically, a preferred route of administration of the humanmonoclonal antibodies of the invention is by topical adminstration,especially directly to the virally induced lesion. Techniques forpreparing topical or transdermal delivery systems containing theantibody of the invention are well known those of skill in the art.

Generally, such systems should utilize components which will notsignificantly impair the biological properties of the antibody, such asthe paratope binding capacity (see, for example, Sciarra and Cutie,Aerosols, in Remington Pharmaceutical Sciences, 18th edition, 1990, pp1694-1712; incorporated by reference). Those of skill in the art canreadily determine the various parameters and conditions for producingantibody-containing compositions without resort to undueexperimentation. Topical compositions include various known mixtureswhich may be applied topically and which allow even spreading of theactive ingredient over the affected area. Accordingly, such topicalcompositions include those pharmaceutically acceptable forms in whichthe compound is applied externally by direct contact with the skinsurface to be treated. Examples of such conventional pharmaceuticalforms of topical formulations include creams, lotions, solutions, gels,ointments and unguents. The formulation may optionally containadditional agents other than the ones of the invention, which arebiologically active or inactive. Such inactive agents includesurfactants, humectants, wetting agents, emulsifiers, and propellantsthat allow even spreading over the affected area.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

The invention also relates to a method for preparing a medicament orpharmaceutical composition comprising the human monoclonal antibodies ofthe invention, the medicament being used for immunotherapy of HSVdisease.

A preferred embodiment of the invention relates to human monoclonalantibodies which neutralize both HSV-1 and -2 whose heavy chainscomprise in CDR3 the polypeptide VAYMLEPTVTAGGLDV (SEQUENCE ID No. 1),and conservative variations of the peptide.

The amino acid sequence of the entire variable region of the heavy chainof the antibody of the invention is shown in SEQUENCE ID No. 2. Alsoencompassed by the present invention are certain amino acid sequencesthat bind to epitopic sequences in glycoprotein D of HSV and conferneutralization of HSV when bound thereto. The term "conservativevariation" or "substitution" as used herein denotes the replacement ofan amino acid residue by another, biologically similar residue. Examplesof conservative variations include the substitution of one hydrophobicresidue such as isoleucine, valine, leucine or methionine for another,or the substitution of one polar residue for another, such as thesubstitution of arginine for lysine, glutamic for aspartic acids, orglutamine for asparagine, and the like. The term "conservativevariation" also includes the use of a substituted amino acid in place ofan unsubstituted parent amino acid provided that antibodies having thesubstituted polypeptide also neutralize HSV. Analogously, anotherpreferred embodiment of the invention relates to polynucleotides whichencode the above noted heavy chain polypeptide and to polynucleotidesequences which are complementary to these polynucleotide sequences.Complementary polynucleotide sequences include those sequences whichhybridize to the polynucleotide sequences of the invention understringent hybridization conditions.

The present invention describes methods for producing novelHSV-neutralizing human monoclonal antibodies. The methods are basedgenerally on the use of combinatorial libraries of antibody moleculeswhich can be produced from a variety of sources, and include naivelibraries, modified libraries, and libraries produced directly fromhuman donors exhibiting an HSV-specific immune response.

The combinatorial library production and manipulation methods have beenextensively described in the literature, and will not be reviewed indetail herein, except for those feature required to make and use uniqueembodiments of the present invention. However, the methods generallyinvolve the use of a filamentous phage (phagemid) surface expressionvector system for cloning and expressing antibody species of thelibrary. Various phagemid cloning systems to produce combinatoriallibraries have been described by others. See, for example thepreparation of combinatorial antibody libraries on phagemids asdescribed by Kang, et al., Proc. Natl. Acad. Sci., USA, 88:4363-4366(1991); Barbas, et al., Proc. Natl. Acad. Sci., USA, 88:7978-7982(1991); Zebedee, et al., Proc. Natl. Acad. Sci., USA, 89:3175-3179(1992); Kang, et al., Proc. Natl. Acad. Sci., USA, 88:11120-11123(1991); Barbas, et al., Proc. Natl. Acad. Sci., USA, 89:4457-4461(1992); and Gram, et al., Proc. Natl. Acad. Sci., USA, 89:3576-3580(1992), which references are hereby incorporated by reference.

In one embodiment, the method involves preparing a phagemid library ofhuman monoclonal antibodies by using donor immune cell messenger RNAfrom HSV-infected donors. The donors can be symptomatic of a HSVinfection, but the donor can also be asymptomatic, as the resultinglibrary contains a substantially higher number of HSV-neutralizing humanmonoclonal antibodies. Additionally, because HSV infection is oftenassociated with other diseases, the patient may optionally presentsubstantial symptoms of one or more other diseases typically associatedwith symptomatic or asymptomatic HSV infection, notably AIDS, asdemonstrated by the library utilized herein.

In another embodiment, the donor is naive relative to a conventionalimmune response to HSV, i.e., the donor is not HSV-infected, and yetantibodies in the donor cross-react with one or more HSV antigens.Alternatively, the library can be synthetic, or can be derived from adonor who has an immune response to other antigens.

The method for producing a human monoclonal antibody generally involves(1) preparing separate H and L chain-encoding gene libraries in cloningvectors using human immunoglobulin genes as a source for the libraries,(2) combining the H and L chain encoding gene libraries into a singledicistronic expression vector capable of expressing and assembling aheterodimeric antibody molecule, (3) expressing the assembledheterodimeric antibody molecule on the surface of a filamentous phageparticle, (4) isolating the surface-expressed phage particle usingimmunoaffinity techniques such as panning of phage particles against apreselected antigen, thereby isolating one or more species of phagemidcontaining particular H and L chain-encoding genes and antibodymolecules that immunoreact with the preselected antigen.

As described herein the Examples, the resulting phagemid library can bemanipulated to increase and/or alter the immunospecificities of themonoclonal antibodies of the library to produce and subsequentlyidentify additional, desirable, human monoclonal antibodies of thepresent invention. For example, the heavy (H) chain and light (L) chainimmunoglobulin molecule encoding genes can be randomly mixed (shuffled)to create new HL pairs in an assembled immunoglobulin molecule.Additionally, either or both the H and L chain encoding genes can bemutagenized in a complementarity determining region (CDR) of thevariable region of the immunoglobulin polypeptide, and subsequentlyscreened for desirable immunoreaction and neutralization capabilities.

In one embodiment, the H and L genes can be cloned into separate,monocistronic expression vectors, referred to as a "binary" systemdescribed further herein. In this method, step (2) above differs in thatthe combining of H and L chain encoding genes occurs by theco-introduction of the two binary plasmids into a single host cell forexpression and assembly of a phagemid having the surface accessibleantibody heterodimer molecule.

In one shuffling embodiment, the shuffling can be accomplished with thebinary expression vectors, each capable of expressing a single heavy orlight chain encoding gene.

In the present methods, the antibody molecules are monoclonal becausethe cloning methods allow for the preparation of clonally pure speciesof antibody producing cell lines. In addition, the monoclonal antibodiesare human because the H and L chain encoding genes are derived fromhuman immunoglobulin producing immune cells, such as spleen, thymus,bone marrow, and the like.

In a method for producing a HSV-neutralizing human monoclonal antibody,it is also required that the resulting antibody library, immunoreactivewith a preselected HSV antigen, be additionally screened for thepresence of antibody species which have the capacity to neutralize HSVin one or more of the assays described herein for determiningneutralization capacity. Thus, a preferred library of antibody moleculesis first produced which binds to an HSV antigen, and then is screenedfor the presence of HSV-neutralizing antibodies as described herein.Additional libraries can be screened from shuffled libraries foradditional HSV-immunoreactive and neutralizing human monoclonalantibodies.

As a further characterization of the present invention the nucleotideand corresponding amino acid residue sequence of the antibody molecule'sH or L chain encoding gene is determined by nucleic acid sequencing. Theprimary amino acid residue sequence information provides essentialinformation regarding the antibody molecule's epitope reactivity.

Sequence comparisons of identified HSV-immunoreactive monoclonalantibody variable chain region sequences are aligned based on sequencehomology, and groups of related antibody molecules are identified inwhich heavy chain or light chain genes share substantial sequencehomology.

An exemplary preparation of a human monoclonal antibody is described inthe Examples. The isolation of a particular vector capable of expressingan antibody of interest involves the introduction of the dicistronicexpression vector into a host cell permissive for expression offilamentous phage genes and the assembly of phage partides. Where thebinary vector system is used, both vectors are introduced in the hostcell. Typically, the host is E. coli. Thereafter, a helper phage genomeis introduced into the host cell containing the immunoglobulinexpression vector(s) to provide the genetic complementation necessary toallow phage particles to be assembled. The resulting host cell iscultured to allow the introduced phage genes and immunoglobulin genes tobe expressed, and for phage particles to be assembled and shed from thehost cell. The shed phage particles are then harvested (collected) fromthe host cell culture media and screened for desirable immunoreactionand neutralization properties. Typically, the harvested particles are"panned" for immunoreaction with a preselected antigen. For example, thepreselected antigen, such as a virus, can be attached directly to asolid phase or indirectly to a solid phase via an antibody specific forthe antigen wherein the antibody is first attached to the solid phasebefore the particles are panned. The strongly immunoreactive particlesare then collected, and individual species of particles are clonallyisolated and further screened for HSV neutralization. Phage whichproduce neutralizing antibodies are selected and used as a source of ahuman HSV neutralizing monoclonal antibody of this invention.

Human monoclonal antibodies of this invention can also be produced byaltering the nucleotide sequence of a polynucleotide sequence thatencodes a heavy or light chain of a monoclonal antibody of thisinvention. For example, by site directed mutagenesis, one can alter thenucleotide sequence of an expression vector and thereby introducechanges in the resulting expressed amino acid residue sequence. One cantake a known polynucleotide and randomly after it by random mutagenesis,reintroduce the altered polynucleotide into an expression system andsubsequently screen the product H:L pair for HSV-neutralizing activity.

Site-directed and random mutagenesis methods are well known in thepolynucleotide arts, and are not to be construed as limiting as methodsfor altering the nucleotide sequence of a subject polynucleotide.

Because an immunoaffinity isolated antibody composition includes phageparticles containing surface antibody, one embodiment involves themanipulation of the resulting cloned genes to truncate theimmunoglobulin-coding gene such that a soluble Fab fragment is secretedby the host E. coli cell containing the phagemid vector rather than theproduction of a phagemid having surface antibody. Thus, the resultingmanipulated cloned immunoglobulin genes produce a soluble Fab which canbe readily characterized in ELISA assays for epitope binding studies, incompetition assays with known anti-HSV antibody molecules, and in HSVneutralization assays. The solubilized Fab provides a reproducible andcomparable antibody preparation for comparative and characterizationstudies.

The preparation of soluble Fab is generally described in theimmunological arts, and can be conducted as described herein in theExamples, or as described by Burton, et al., Proc. Natl. Acad. Sci.,USA, 88:10134-10137 (1991). The preparation of human monoclonalantibodies of this invention depends, in one embodiment, on the cloningand expression vectors used to prepare the combinatorial antibodylibraries described herein. The cloned immunoglobulin heavy and lightchain genes can be shuttled between lambda vectors, phagemid vectors andplasmid vectors at various stages of the methods described herein.

The phagemid vectors produce fusion proteins that are expressed on thesurface of an assembled filamentous phage particle. A preferred phagemidvector of the present invention is a recombinant DNA (rDNA) moleculecontaining a nucleotide sequence that codes for and is capable ofexpressing a fusion polypeptide containing, in the direction of amino-to carboxy-terminus, (1) a prokaryotic secretion signal domain, (2) aheterologous polypeptide defining an immunoglobulin heavy or light chainvariable region, and (3) a filamentous phage membrane anchor domain. Thevector includes DNA expression control sequences for expressing thefusion polypeptide, preferably prokaryotic control sequences.

The filamentous phage membrane anchor is preferably a domain of thecpIII or cpVIII coat protein capable of associating with the matrix of afilamentous phage particle, thereby incorporating the fusion polypeptideonto the phage surface. The secretion signal is a leader peptide domainof a protein that targets the protein to the periplasmic membrane ofgram negative bacteria. A preferred secretion signal is a pelB secretionsignal. The predicted amino acid residue sequences of the secretionsignal domain from two pelB gene product variants from Erwinia carotovaare described in Lei, et al., Nature, 331:543-546 (1988).

The leader sequence of the pelB protein has previously been used as asecretion signal for fusion proteins. Better, et al., Science,240:1041-1043 (1988); Sastry, et al., Proc. Nat. Acad. Sci., USA,86:5728-5732 (1989); and Mullinax, et al., Proc. Natl. Acad. Sci., USA,87:8095-8099 (1990). Amino add residue sequences for other secretionsignal polypeptide domains from E. coli useful in this invention asdescribed in Oliver, Escherichia coli and Salmonella typhimurium,Neidhard, F. C. (ed.), American Society for Microbiology, Washington,D.C., 1:56-69 (1987).

Preferred membrane anchors for the vector are obtainable fromfilamentous phage M13, f1, fd, and equivalent filamentous phage.Preferred membrane anchor domains are found in the coat proteins encodedby gene III and gene VIII. The membrane anchor domain of a filamentousphage coat protein is a portion of the carboxy terminal region of thecoat protein and includes a region of hydrophobic amino acid residuesfor spanning a lipid bilayer membrane, and a region of charged amino addresidues normally found at the cytoplasmic face of the membrane andextending away from the membrane.

In the phage f1, gene VIII coat protein's membrane spanning regioncomprises residue Trp-26 through Lys-40, and the cytoplasmic regioncomprises the carboxy-terminal 11 residues from 41 to 52 (Ohkawa, etal., J. Biol. Chem., 256:9951-9958 (1981)). An exemplary membrane anchorwould consist of residues 26 to 40 of cpVIII. Thus, the amino addresidue sequence of a preferred membrane anchor domain is derived fromthe M13 filamentous phage gene VIII coat protein (also designated cpVIIIor CP 8). Gene VIII coat protein is present on a mature filamentousphage over the majority of the phage particle with typically about 2500to 3000 copies of the coat protein.

In addition, the amino acid residue sequence of another preferredmembrane anchor domain is derived from the M13 filamentous phage geneIII coat protein (also designated cpIII). Gene III coat protein ispresent on a mature filamentous phage at one end of the phage particlewith typically about 4 to 6 copies of the coat protein.

For detailed descriptions of the structure of filamentous phageparticles, their coat proteins and particle assembly, see the reviews byRached, et al., Microbiol. Rev., 50:401-427 (1986); and Model, et al.,in "The Bacteriophages: Vol. 2", R. Calendar, ed. Plenum Publishing Co.,pp. 375-456 (1988).

DNA expression control sequences comprise a set of DNA expressionsignals for expressing a structural gene product and include both 5' and3' elements, as is well known, operatively linked to the cistron suchthat the cistron is able to express a structural gene product. The 5'control sequences define a promoter for initiating transcription and aribosome binding site operatively linked at the 5' terminus of theupstream translatable DNA sequence.

To achieve high levels of gene expression in E. coli, it is necessary touse not only strong promoters to generate large quantities of mRNA, butalso ribosome binding sites to ensure that the mRNA is efficientlytranslated. In E. coli, the ribosome binding site includes an initiationcodon (AUG) and a sequence 3-9 nucleotides long located 3-11 nucleotidesupstream from the initiation codon (Shine, et al., Nature, 254:34(1975). The sequence, AGGAGGU, which is called the Shine-Dalgarno (SD)sequence, is complementary to the 3' end of E. coil 16S rRNA. Binding ofthe ribosome to mRNA and the sequence at the 3' end of the mRNA can beaffected by several factors:

(i) The degree of complementarity between the SD sequence and 3' end ofthe 16S rRNA.

(ii) The spacing and possibly the DNA sequence lying between the SDsequence and the AUG. Roberts, et al., Proc. Natl. Acad. Sci., USA,76:760, (1979a); Roberts, et al., Proc. Natl. Acad. Sci. USA, 76:5596(1979b); Guarente, et al., Science, 209:1428 (1980); and Guarente, etal., Cell, 20:543 (1980). Optimization is achieved by measuring thelevel of expression of genes in plasmids in which this spacing issystematically aftered. Comparison of different mRNAs shows that thereare statistically preferred sequences from positions -20 to +13 (wherethe A of the AUG is position 0). Gold, et al., Annu. Rev. Microbiol.,35:365 (1981). Leader sequences have been shown to influence translationdramatically. Roberts, et al., 1979 a, b supra.

(iii) The nucleotide sequence following the AUG, which affects ribosomebinding. Taniguchi, et al., J. Mol. Biol., 118:533 (1978). The 3'control sequences define at least one termination (stop) codon in framewith and operatively linked to the heterologous fusion polypeptide.

In preferred embodiments, the vector utilized includes a prokaryoticorigin of replication or replicon, i.e., a DNA sequence having theability to direct autonomous replication and maintenance of therecombinant DNA molecule extra chromosomally in a prokaryotic host cell,such as a bacterial host cell, transformed therewith. Such origins ofreplication are well known in the art. Preferred origins of replicationare those that are efficient in the host organism. A preferred host cellis E. coli. For use of a vector in E. coli, a preferred origin ofreplication is ColE1 found in pBR322 and a variety of other commonplasmids. Also preferred is the p15A origin of replication found onpACYC and its derivatives. The ColE1 and p15A replicon have beenextensively utilized in molecular biology, are available on a variety ofplasmids and are described at least by Sambrook, et al., in "MolecularCloning: a Laboratory Manual", 2nd edition, Cold Spring HarborLaboratory Press (1989).

The ColE1 and p15A replicons are particularly preferred for use in oneembodiment of the present invention where two "binary" plasmids areutilized because they each have the ability to direct the replication ofplasmid in E. coil while the other replicon is present in a secondplasmid in the same E. coli cell.

In other words, ColE1 and p15A are non-interfering replicons that allowthe maintenance of two plasmids in the same host (see, for example,Sambrook, et al., supar, at pages 1.3-1.4). This feature is particularlyimportant in the binary vectors embodiment of the present inventionbecause a single host cell permissive for phage replication must supportthe independent and simultaneous replication of two separate vectors,namely a first vector for expressing a heavy chain polypeptide, and asecond vector for expressing a light chain polypeptide.

In addition, those embodiments that include a prokaryotic replicon canalso include a gene whose expression confers a selective advantage, suchas drug resistance, to a bacterial host transformed therewith. Typicalbacterial drug resistance genes are those that confer resistance toampicillin, tetracycline, neomycin/kanamycin or cholamphenicol. Vectorstypically also contain convenient restriction sites for insertion oftranslatable DNA sequences. Exemplary vectors are the plasmids pUC8,pUC9, pBR322, and pBR329 available from BioRad Laboratories, (Richmond,Calif.) and pPL and pKK223 available from Pharmacia, (Piscataway, N.J.).

A vector for expression of a monoclonal antibody of the invention on thesurface of a filamentous phage particle is a recombinant DNA (rDNA)molecule adapted for receiving and expressing translatable first andsecond DNA sequences in the form of first and second polypeptideswherein one of the polypeptides is fused to a filamentous phage coatprotein membrane anchor. That is, at least one of the polypeptides is afusion polypeptide containing a filamentous phage membrane anchordomain, a prokaryotic secretion signal domain, and an immunoglobulinheavy or light chain variable domain.

A DNA expression vector for expressing a heterodimeric antibody moleculeprovides a system for independently cloning (inserting) the twotranslatable DNA sequences into two separate cassettes present in thevector, to form two separate cistrons for expressing the first andsecond polypeptides of the antibody molecule, or the ligand bindingportions of the polypeptides that comprise the antibody molecule (i.e.,the H and L variable regions of an immunoglobulin molecule). The DNAexpression vector for expressing two cistrons is referred to as adicistronic expression vector.

The vector comprises a first cassette that includes upstream anddownstream translatable DNA sequences operatively linked via a sequenceof nucleotides adapted for directional ligation to an insert DNA. Theupstream translatable sequence encodes the secretion signal as definedherein. The downstream translatable sequence encodes the filamentousphage membrane anchor as defined herein. The cassette preferablyincludes DNA expression control sequences for expressing the receptorpolypeptide that is produced when an insert translatable DNA sequence(insert DNA) is directionally inserted into the cassette via thesequence of nucleotides adapted for directional ligation. Thefilamentous phage membrane anchor is preferably a domain of the cpIII orcpVIII coat protein capable of binding the matrix of a filamentous phageparticle, thereby incorporating the fusion polypeptide onto the phagesurface.

The receptor expressing vector also contains a second cassette forexpressing a second receptor polypeptide. The second cassette includes asecond translatable DNA sequence that encodes a secretion signal, asdefined herein, operatively linked at its 3' terminus via a sequence ofnucleotides adapted for directional ligation to a downstream DNAsequence of the vector that typically defines at least one stop codon inthe reading frame of the cassette. The second translatable DNA sequenceis operatively linked at its 5' terminus to DNA expression controlsequences forming the 5' elements. The second cassette is capable, uponinsertion of a translatable DNA sequence (insert DNA), of expressing thesecond fusion polypeptide comprising a receptor of the secretion signalwith a polypeptide coded by the insert DNA.

An upstream translatable DNA sequence encodes a prokaryotic secretionsignal as described earlier. The upstream translatable DNA sequenceencoding the pelB secretion signal is a preferred DNA sequence forinclusion in a receptor expression vector. A downstream translatable DNAsequence encodes a filamentous phage membrane anchor as describedearlier. Thus, a downstream translatable DNA sequence encodes an aminoacid residue sequence that corresponds, and preferably is identical, tothe membrane anchor domain of either a filamentous phage gene III orgene VIII coat polypeptide.

A cassette in a DNA expression vector of this invention is the region ofthe vector that forms, upon insertion of a translatable DNA sequence(insert DNA), a sequence of nucleotides capable of expressing, in anappropriate host, a fusion polypeptide. The expression-competentsequence of nucleotides is referred to as a cistron. Thus, the cassettecomprises DNA expression control elements operatively linked to theupstream and downstream translatable DNA sequences. A cistron is formedwhen a translatable DNA sequence is directionally inserted(directionally ligated) between the upstream and downstream sequencesvia the sequence of nucleotides adapted for that purpose. The resultingthree translatable DNA sequences, namely the upstream, the inserted andthe downstream sequences, are all operatively linked in the same readingframe.

Thus, a DNA expression vector for expressing an antibody moleculeprovides a system for cloning translatable DNA sequences into thecassette portions of the vector to produce cistrons capable ofexpressing the first and second polypeptides, i.e., the heavy and lightchains of a monoclonal antibody.

As used herein, the term "vector" refers to a nucleic acid moleculecapable of transporting between different genetic environments anothernucleic acid to which it has been operatively linked. Preferred vectorsare those capable of autonomous replication and expression of structuralgene products present in the DNA segments to which they are operativelylinked. Vectors, therefore, preferably contain the replicons andselectable markers described earlier.

As used herein with regard to DNA sequences or segments, the phrase"operatively linked" means the sequences or segments have beencovalently joined, preferably by conventional phosphodiester bonds, intoone strand of DNA, whether in single or double stranded form. The choiceof vector to which transcription unit or a cassette of this invention isoperatively linked depends directly, as is well known in the art, on thefunctional properties desired, e.g., vector replication and proteinexpression, and the host cell to be transformed, these being limitationsinherent in the art of constructing recombinant DNA molecules.

A sequence of nucleotides adapted for directional ligation, i.e., apolylinker, is a region of the DNA expression vector that (1)operatively links for replication and transport the upstream anddownstream translatable DNA sequences and (2) provides a site or meansfor directional ligation of a DNA sequence into the vector. Typically, adirectional polylinker is a sequence of nucleotides that defines two ormore restriction endonuclease recognition sequences, or restrictionsites. Upon restriction cleavage, the two sites yield cohesive terminito which a translatable DNA sequence can be ligated to the DNAexpression vector. Preferably, the two restriction sites provide, uponrestriction cleavage, cohesive termini that are non-complementary andthereby permit directional insertion of a translatable DNA sequence intothe cassette. In one embodi- ment, the directional ligation means isprovided by nucleotides present in the upstream translatable DNAsequence, downstream translatable DNA sequence, or both. In anotherembodiment, the sequence of nucleotides adapted for directional ligationcomprises a sequence of nucleotides that defines multiple directionalcloning means. Where the sequence of nucleotides adapted for directionalligation defines numerous restriction sites, it is referred to as amultiple cloning site.

In a preferred embodiment, a DNA expression vector is designed forconvenient manipulation in the form of a filamentous phage particleencapsulating a genome according to the teachings of the presentinvention. In this embodiment, a DNA expression vector further containsa nucleotide sequence that defines a filamentous phage origin ofreplication such that the vector, upon presentation of the appropriategenetic complementation, can replicate as a filamentous phage in singlestranded replicative form and be packaged into filamentous phageparticles. This feature provides the ability of the DNA expressionvector to be packaged into phage particles for subsequent segregation ofthe particle, and vector contained therein, away from other particlesthat comprise a population of phage particles.

A filamentous phage origin of replication is a region of the phagegenome, as is well known, that defines sites for initiation ofreplication, termination of replication and packaging of the replicativeform produced by replication (see, for example, Rasched, et al.,Microbiol. Rev., 50:401-427 (1986); and Horiuchi, J. Mol. Biol.,188:215-223 (1986)).

A preferred filamentous phage origin of replication for use in thepresent invention is an M13, f1 or fd phage origin of replication(Short, et al., Nucl. Acids Res., 16:7583-7600 (1988)). Preferred DNAexpression vectors for cloning and expression a human monoclonalantibody of this invention are the dicistronic expression vectorspCOMB8, pCOMB2-8, pCOMB3, pCOMB2-3 and pCOMB2-3', described herein.

A particularly preferred vector of the present invention includes apolynucleotide sequence that encodes a heavy or light chain variableregion of a human monoclonal antibody of the present invention.Particularly preferred are vectors that include a nucleotide sequencethat encodes a heavy chain CDR3 amino acid residue sequence shown inSEQUENCE ID No.1 that encodes a heavy chain having the bindingspecificity of those sequences shown in SEQUENCE ID No. 1 or thatencodes a heavy chain CDR3 region having conservative substitutionsrelative to a sequence shown in SEQUENCE ID No. 1, and complementarypolynucleotide sequences thereto. The entire heavy chain amino acidresidue sequence for the preferred antibody of the invention is shown inSEQUENCE ID No. 2.

Insofar as polynucleotides are component parts of a DNA expressionvector for producing a human monoclonal antibody heavy or light chainimmunoglobulin variable region amino acid residue sequence, theinvention also contemplates isolated polynucleotides that encode suchheavy or light chain sequences.

It is to be understood that, due to the genetic code and its attendantredundancies, numerous polynucleotide sequences can be designed thatencode a contemplated heavy or light chain immunoglobulin variableregion amino acid residue sequence. Thus, the invention contemplatessuch alternate polynucleotide sequences incorporating the features ofthe redundancy of the genetic code.

Insofar as the expression vector for producing a human monoclonalantibody of this invention is carried in a host cell compatible withexpression of the antibody, the invention contemplates a host cellcontaining a vector or polynucleotide of this invention. A preferredhost cell is E. coli, as described herein.

An E. coli culture containing a preferred expression vector thatproduces a human monoclonal antibody of this invention was depositedpursuant to Budapest Treaty requirements with the American Type CultureCollection (ATCC), Rockville, Md., as described herein.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that various changes and modifications can bemade without departing from the spirit or scope of the invention.

EXAMPLE 1 CONSTRUCTION OF A DICISTRONIC EXPRESSION VECTOR FOR PRODUCINGA HETERODIMERIC RECEPTOR ON PHAGE PARTICLES

To obtain a vector system for generating a large number of Fab antibodyfragments that can be screened directly, expression libraries inbacteriophage Lambda have previously been constructed as described inHuse, et al (Science, 246:1275-1281, 1989). However, these systems didnot contain design features that provide for the expressed Fab to betargeted to the surface of a filamentous phage particle as described byBarbas, et al (Proc. Natl. Acad. Sci. USA, 33:7978-7982, 1991).

The main criterion used in choosing a vector system was the necessity ofgenerating the largest number of Fab fragments which could be screeneddirectly. Bacteriophage Lambda was selected as the starting point todevelop an expression vector for three reasons. First, in vitropackaging of phage DNA was the most efficient method of reintroducingDNA into host cells. Second, it was possible to detect proteinexpression at the level of single phage plaques. Finally, the screeningof phage libraries typically involved less difficulty with nonspecificbinding. The alternative, plasmid cloning vectors, are only advantageousin the analysis of clones after they have been identified. Thisadvantage was not lost in the present system because of the use of adicistronic expression vector such as pCombVIII, thereby permitting aplasmid containing the heavy chain, light chain, or Fab expressinginserts to be excised.

a. Construction of Dicistronic Expression Vector pCOMB

(i) Preparation of Lambda Zap™ II

Lambda Zap™ II is a derivative of the original Lambda Zap (ATCC #40,298)that maintains all of the characteristics of the original Lambda Zapincluding 6 unique cloning sites, fusion protein expression, and theability to rapidly excise the insert in the form of a phagemid(Bluescript SK-), but lacks the SAM 100 mutation, allowing growth onmany Non-Sup F strains, including XL1-Blue. The Lambda Zaps™ II wasconstructed as described in Short, et al., (Nuc. Acids Res.,16:7583-7600, 1988), by replacing the lambda S gene contained in a 4254base pair (bp) DNA fragment produced by digesting lambda Zap with therestriction enzyme Nco I. This 4254 bp DNA fragment was replaced withthe 4254 bp DNA fragment containing the Lambda S gene isolated fromLambda gt10 (ATCC #40,179) after digesting the vector with therestriction enzyme Nco I. The 4254 bp DNA fragment isolated from lambdagt10 was ligated into the original lambda Zap vector using T4 DNA ligaseand standard protocols such as those described in Current Protocols inMolecular Biology, Ausubel, et al., eds., John Wiley and Sons, N.Y.,1987, to form Lambda Zap™ II.

(ii) Preparation of Lambda Hc2

To express a plurality of V_(H) -coding DNA homologs in an E. coli hostcell, a vector designated Lambda Hc2 was constructed. The vectorprovided the following: the capacity to place the V_(H) -coding DNAhomologs in the proper reading frame; a ribosome binding site asdescribed by Shine, et al., (Nature, 254:34, 1975); a leader sequencedirecting the expressed protein to the periplasmic space designated thepelB secretion signal; a polynucleotide sequence that coded for a knownepitope (epitope tag); and also a polynucleotide that coded for a spacerprotein between the V_(H) -coding DNA homolog and the polynucleotidecoding for the epitope tag. Lambda Hc2 has been previously described byHuse, et al., (Science, 246:1274-1281, 1989).

To prepare Lambda Hc2, a synthetic DNA sequence containing all of theabove features was constructed by designing single strandedpolynucleotide segments of 20-40 bases that would hybridize to eachother and form the double stranded synthetic DNA sequence. Theindividual single-stranded polynucleotide segments are shown in Table 1.

Polynucleotides N2, N3, N9-4, N11, N10-5, N6, N7 and N8 (Table 1) werekinased by adding 1 μl of each polynucleotide (0.1 μg/μl) and 20 unitsof T₄ polynucleotide kinase to a solution containing 70 mM Tris-HCl, pH7.6, 10 mM MgCl₂, 5 mM dithiothreitol (DTT), 10 mM beta-mercaptoethanol,500 micro-grams per milliliter μg/ml) bovine serum albumin (BSA). Thesolution was maintained at 37 degrees Centigrade (37° C.) for 30 minutesand the reaction stopped by maintaining the solution at 65° C. for 10minutes. The two end polynucleotides, 20 mg of polynucleotides N1 andpolynucleotides N12, were added to the above kinasing reaction solutiontogether with 1/10 volume of a solution containing 20.0 mM Tris-HCl, pH7.4, 2.0 mM MgCl₂ and 50.0 mM NaCl. This solution was heated to 70° C.for 5 minutes and allowed to cool to room temperature, approximately 25°C., over 1.5 hours in a 500 ml beaker of water. During this time periodall 10 polynucleotides annealed to form a double stranded synthetic DNAinsert. The individual polynucleotides were covalently linked to eachother to stabilize the synthetic DNA insert by adding 40 μl of the abovereaction to a solution containing 50 mM Tris-HCl, pH 7.5, 7 mM MgCl₂, 1mM DTT, 1 mM adenosine triphosphate (ATP) and 10 units of T4 DNA ligase.This solution was maintained at 37° C. for 30 minutes and then the T4DNA ligase was inactivated by maintaining the solution at 65° C. for 10minutes. The end polynucleotides were kinased by mixing 52 μl of theabove reaction, 4 μl of a solution containing 10 mM ATP and 5 units ofT4 polynucleotide kinase. This solution was maintained at 37° C. for 30minutes and then the T4 polynucleotide kinase was inactivated bymaintaining the solution at 65° C. for 10 minutes.

                  TABLE 1                                                         ______________________________________                                        N1)   5' GGCCGCAAATTCTATTTCAAGGAGACAGTCAT 3'                                                   (SEQUENCE ID NO. 3)                                          N2)          5' AATGAAATACCTATTGCCTACGGCAGCCGCTGGATT 3'                                       (SEQUENCE ID NO. 4)                                           N3)          5' GTTATTACTCGCTGCCCAACCAGCCATGGCCC 3'                                          (SEQUENCE ID NO. 5)                                            N6)          5' CAGTTTCACCTGGGCCATGGCTGGTTGGG 3'                                            (SEQUENCE ID NO. 6)                                             N7)          5'CAGCGAGTAATAACAATCCAGCGGCTGCCGTAGGCAATAG3'                                  (SEQUENCE ID NO. 7)                                              N8)          5' GTATTTCATTATGACTGTCTCCTTGAAATAGAATTTGC 3'                                 (SEQUENCE ID NO. 8)                                               N9-4)      5'AGGTGAAACTGCTCGAGATTTCTAGACTAGTTACCCGTAC3'                                  (SEQUENGE ID NO. 9)                                                N10-5)                                                                                  5' CGGAACGTCGTACGGGTAACTAGTCTAGAAATCTCGAG 3'                                  (SEQUENCE ID NO. 10)                                                N11)        5' GACGTTCCGGACTACGGTTCTTAATAGAATTCG 3'                                    (SEQUENCE ID NO. 11)                                                 N12)        5' TCGACGAATTCTATTAAGAACCGTAGTC 3'                                        (SEQUENCE ID NO. 12)                                                  ______________________________________                                    

The completed synthetic DNA insert was ligated directly into the LambdaZap™ II vector described in Example 1 a(i) that had been previouslydigested with the restriction enzymes, Not I and Xho I. The ligationmixture was packaged according to the manufacture's instructions usingGigapack II Gold packing extract available from Stratagene, La Jolla,Calif. The packaged ligation mixture was plated on XL1-Blue cells(Stratagene). Individual Lambda plaques were cored and the insertsexcised according to the in vivo excision protocol for Lambda Zap™ IIprovided by the manufacturer (Stratagene). This in vivo excisionprotocol moved the cloned insert from the Lambda Hc2 vector into aphagemid vector to allow for easy manipulation and sequencing. Theaccuracy of the above cloning steps was confirmed by sequencing theinsert using the Sanger dideoxy method described in by Sanger, et al.,(Proc. Natl. Aced. Sci. USA, 74:5463-5467, 1977), and using themanufacture's instructions in the AMV Reverse Transcriptase ³⁵ S-ATPsequencing kit (Stratagene).

(iii) Preparation of Lambda Lc2

To express a plurality of V_(L) -coding DNA homologs in an E.coli hostcell, a vector designated Lambda Lc2 was constructed having the capacityto place the V_(L) -coding DNA homologs in the proper reading frame,provided a ribosome binding site as described by Shine, et al., (Nature,254:34, 1975), provided the pelB gene leader sequence secretion signalthat has been previously used to successfully secrete Fab fragments inE. coli by Lei, et aL (J. Bac., 169:4379, 1987) and Better, et al.,(Science, 240: 1041, 1988), and also provided a polynucleotidecontaining a restriction endonuclease site for cloning. Lambda Lc2 hasbeen previously described by Huse, et al., (Science, 246:1275-1281,1989).

A synthetic DNA sequence containing all of the above features wasconstructed by designing single stranded polynucleotide segments of20-60 bases that would hybridize to each other and form the doublestranded synthetic DNA. The sequence of each individual single-strandedpolynucleotide segment (1-8) within the double stranded synthetic DNAsequence is shown in Table 2.

Polynucleotides shown in Table 2 were kinased by adding 1 μl (0.1 μg/μl)of each polynucleotide and 20 units of T₄ polynucleotide kinase to asolution containing 70 mM Tris-HCl, pH 7.6, 10 mM MgCl, 5 mM DTT, 10 mMbeta-mercaptoethanol, 500 mg/ml of BSA. The solution was maintained at37° C. for 30 minutes and the reaction stopped by maintaining thesolution at 65° C. for 10 minutes. The 20 ng each of the two endpolynucleotides, 01 and 08, were added to the above kinasing reactionsolution together with 1/10 volume of a solution containing 20.0 mMTris-HCl, pH 7.4, 2.0 mM MgCl and 15.0 mM sodium chloride (NaCl). Thissolution was heated to 70° C. for 5 minutes and allowed to cool to roomtemperature, approximately 25° C., over 1.5 hours in a 500 ml beaker ofwater. During this time period all 8 polynucleotides annealed to formthe double stranded synthetic DNA insert shown in FIG. 3. The individualpolynucleotides were covalently linked to each other to stabilize thesynthetic DNA insert by adding 40 μl of the above reaction to a solutioncontaining 50 ml Tris-HCl, pH 7.5, 7 ml MgCl, 1 mm DTT, 1 mm ATP and 10units of T4 DNA ligase. This solution was maintained at 37° C. for 30minutes and then the T4 DNA ligase was inactivated by maintaining thesolution at 65° C. for 10 minutes. The end polynucleotides were kinasedby mixing 52 μl of the above reaction, 4 μl of a solution containing 10mM ATP and 5 units of T4 polynucleotide kinase. This solution wasmaintained at 37° C. for 30 minutes and then the T4 polynucleotidekinase was inactivated by maintaining the solution at 65° C. for 10minutes.

                                      TABLE 2                                     __________________________________________________________________________      5' TGAATTCTAAACTAGTCGCCAAGGAGACAGTCAT 3'                                           (SEQUENCE ID NO. 13)                                                      5' AATGAAATACCTATTGCCTACGGCAGCCGCTGGATT 3'                                       (SEQUENCE ID NO. 14)                                                       5' GTTATTACTCGCTGCCCAACCAGCCATGGCC 3'                                           (SEQUENCE ID NO. 15)                                                        5' GAGCTCGTCAGTTCTAGAGTTAAGCGGCCG 3'                                           (SEQUENCE ID NO. 16)                                                         5' CTATTTCATTATGACTGTCTCCTTGGCGACTAGTTTAGAATTCAAGCT 3'                        (SEQUENCE ID NO. 17)                                                          5' CAGCGAGTAATAACAATCCAGCGGCTGCCGTAGGCAATAG 3'                               (SEQUENCE ID NO. 18)                                                           5' TGACGAGCTCGGCCATGGCTGGTTGGG 3'                                            (SEQUENCE ID NO. 19)                                                           5' TCGACGGCCGCTTAACTCTAGAAC 3'                                               (SEQUENCE ID NO. 20)                                                        __________________________________________________________________________

The completed synthetic DNA insert was ligated directly into the LambdaZap™ II vector described in Example 1(a) (i) that had been previouslydigested with the restriction enzymes Sac I and Xho I. The ligationmixture was packaged according to the manufacture's instructions usingGigapack II Gold packing extract (Stratagene). The packaged ligationmixture was plated on XL1-Blue cells (Stratagene). Individual Lambdaplaques were cored and the inserts excised according to the in vivoexcision protocol for Lambda Zap™ II provided by the manufacturer(Stratagene). This in vivo excision protocol moved the cloned insertfrom the Lambda Lc2 vector into a plasmid phagemid vector allow for easymanipulation and sequendng. The accuracy of the above cloning steps wasconfirmed by sequencing the insert using the manufacture's instructionsin the AMV Reverse Transcriptase ³⁵ S-dATP sequencing kit (Stratagene).

A preferred vector for use in this invention, designated Lambda Lc3, isa derivative of Lambda Lc2 prepared above. Lambda Lc2 contains a Spe Irestriction site (ACTAGT) located 3' to the EcoR I restriction site and5' to the Shine-Dalgarno ribosome binding site. A Spe I restriction siteis also present in Lambda Hc2. A combinatorial vector, designated pComb,was constructed by combining portions of Lambda Hc2 and Lc2 together asdescribed in Example 1a(iv) below. The resultant combinatorial pCombvector contained two Spe I restriction sites, one provided by Lambda Hc2and one provided by Lambda Lc2, with an EcoR I site in between. Despitethe presence of two Spe I restriction sites, DNA homologs having Spe Iand EcoR I cohesive termini were successfully directionally ligated intoa pComb expression vector previously digested with Spe I and EcoR I. Theproximity of the EcoR I restriction site to the 3' Spe I site, providedby the Lc2 vector, inhibited the complete digestion of the 3' Spe Isite. Thus, digesting pComb with Spe I and EcoR I did not result inremoval of the EcoR I site between the two Spe I sites.

The presence of a second Spe I restriction site may be undesirable forligations into a pComb vector digested only with Spe I as the regionbetween the two sites would be eliminated. Therefore, a derivative ofLambda Lc2 lacking the second or 3' Spe I site, designated Lambda Lc3,is produced by first digesting Lambda Lc2 with Spe I to form alinearized vector. The ends are filled in to form blunt ends which areligated together to result in Lambda Lc3 lacking a Spe I site. LambdaLc3 is a preferred vector for use in constructing a combinatorial vectoras described below.

(iv) Preparation of pComb

Phagemids were excised from the expression vectors lambda Hc2 or LambdaLc2 using an in vivo excision protocol described above. Double strandedDNA was prepared from the phagemid-containing cells according to themethods described by Holmes, et al., (Anal. Biochem., 114:193, 1981).The phagemids resulting from in vivo excision contained the samenucleotide sequences for antibody fragment cloning and expression as didthe parent vectors, and are designated phagemid Hc2 and Lc2,corresponding to Lambda Hc2 and Lc2, respectively.

For the construction of combinatorial phagemid vector pComb, produced bycombining portions of phagemid Hc2 and phagemid Lc2, phagemid Hc2 wasfirst digested with Sac I to remove the restriction site located 5' tothe LacZ promoter. The linearized phagemid was then blunt ended with T4polymerase and ligated to result in a Hc2 phagemid lacking a Sac I site.The modified Hc2 phagemid and the Lc2 phagemid were then separatelyrestriction digested with Sca I and EcoR I to result in a Hc2 fragmenthaving from 5' to 3' Sca I, not I Xho I, Spe I and EcoR I restrictionsites and a Lc2 fragment having from 5' to 3' EcoR I, Sac I, Xba I andSac I restriction sites. The linearized phagemids were then ligatedtogether at their respective cohesive ends to form pComb, a circularizedphagemid having a linear arrangement of restriction sites of Not I, XhoI, Spe I, EcoR I, Sac I, Xba I, Apa I and Sca I. The ligated phagemidvector was then inserted into an appropriate bacterial host andtransformants were selected on the antibiotic ampicillin.

Selected ampicillin resistant transformants were screened for thepresence of two Not I sites. The resulting ampicillin resistantcombinatorial phagemid vector was designated pComb. The resultantcombinatorial vector, pComb, consisted of a DNA molecule having twocassettes to express two fusion proteins and having nucleotide residuesequences for the following operatively linked elements listed in a 5'to 3' direction: a first cassette consisting of an inducible LacZpromoter upstream from the LacZ gene; a Not I restriction site; aribosome binding site; a pelB leader; a spacer; a cloning regionbordered by a 5' Xho and 3' Spe I restriction site; a decapeptide tagfollowed by expression control stop sequences; an EcoR I restrictionsite located 5' to a second cassette consisting of an expression controlribosome binding site; a pelB leader; a spacer region; a cloning regionbordered by a 5' Sac I and a 3' Xba I restriction site followed byexpression control stop sequences and a second Not I restriction site.

A preferred combinatorial vector designated pComb3, is constructed bycombining portions of phagemid Hc2 and phagemid Lc3 as described abovefor preparing pComb. The resultant combinatorial vector, pComb3,consists of a DNA molecule having two cassettes identical to pComb toexpress two fusion proteins identically to pComb except that a secondSpe I restriction site in the second cassette is eliminated.

b. Construction of Vector pCombIII for Expressing Fusion Proteins Havinga Bacteriophage Coat Protein Membrane Anchor

Because of the multiple endonuclease restriction cloning sites, thepComb phagemid expression vector prepared above is a useful cloningvehicle for modification for the preparation of an expression vector ofthis invention. To that end, pComb is digested with EcoR I and Spe Ifollowed by phosphatase treatment to produce linearized pComb.

(ii) Preparation of pCombIII

A separate phagemid expression vector was constructed using sequencesencoding bacteriophage cpIII membrane anchor domain. A PCR productdefining the DNA sequence encoding the filamentous phage coat protein,cpIII, membrane anchor containing a LacZ promotor region sequence 3' tothe membrane anchor for expression of the light chain and Spe I and EcoRI cohesive termini was prepared from M13mp 18, a commercially availablebacteriophage vector (Pharmacia, Piscataway, N.J.).

To prepare a modified cpIII, replicative form DNA from M13mp18 was firstisolated. Briefly, into 2 ml of LB (Luria-Bertani medium), 50 μl of aculture of a bacterial strain carrying an F' episome (JM107, JM109 orTG1) were admixed with a one tenth suspension of bacteriophage particlesderived from a single plaque. The admixture was incubated for 4 to 5hours at 37° C. with constant agitation. The admixture was thencentrifuged at 12,000×g for 5 minutes to pellet the infected bacteria.After the supernatant was removed, the pellet was resuspended byvigorous vortexing in 100 μl of ice-cold solution I. Solution I wasprepared by admixing 50 mM glucose, 10 mM EDTA (disodiumethylenediaminetetraacetic acid) and 25 mM Tris-HCl at pH 8.0, andautoclaving for 15 minutes.

To the bacterial suspension, 200 μl of freshly prepared Solution II wasadmixed and the tube was rapidly inverted five times. Solution II wasprepared by admixing 0.2 N NaOH and 1% SDS. To the bacterial suspension,150 μl of ice-cold Solution III was admixed and the tube was vortexedgently in an inverted position for 10 seconds to disperse Solution IIIthrough the viscous bacterial lysate. Solution III was prepared byadmixing 60 ml of 5 M potassium acetate, 11.5 ml of glacial acetic acidand 28.5 ml of water The resultant bacterial lysate was then stored onice for 5 minutes followed by centrifugation at 12,000×g for 5 minutesat 4° C. in a microfuge. The resultant supernatant was recovered andtransferred to a new tube. To the supernatant was added an equal volumeof phenol/chloroform and the admixture was vortexed. The admixture wasthen centrifuged at 12,000×g for 2 minutes in a microfuge. The resultantsupernatant was transferred to a new tube and the double--strandedbacteriophage DNA was precipitated with 2 volumes of ethanol at roomtemperature. After allowing the admixture to stand at room temperaturefor 2 minutes, the admixture was centrifuged to pellet the DNA. Thesupernatant was removed and the pelleted replicative form DNA wasresuspended in 25 μl of Tris-HCl at pH 7.6, and 10 mM EDTA (TE).

The double-stranded M13mp 18 replicative form DNA was then used as atemplate for isolating the gene encoding the membrane anchor domain atcpIII. M13mp 18 replicative form DNA was prepared as described above andused as a template for two PCR amplifications for construction of a DNAfragment consisting of the mature gene for cpIII membrane anchor domainlocated 5' to a sequence encoding the LacZ promoter, operator andcap-binding site for controlling light chain expression. The restrictionsites, Spe I and EcoR I, were created in the amplification reactions andwere located at the 5' and 3' ends of the fragment respectively. Theprocedure for creating this fragment by combining the products of twoseparate PCR amplifications is described below.

The primer pair, G-3(F) (SEQUENCE ID No. 21) and G-3(B) (SEQUENCE ID No.22) listed in Table 3, was used in the first PCR reaction as performedabove to amplify the cpIII membrane anchor gene and incorporate Spe Iand Nhe I restriction sites into the fragment. For the PCR reaction, 2μl containing 1 ng of M13mp 18 replicative form DNA were admixed with 10μl of 10× PCR buffer purchased commercially (Promega Biotech, Madison,Wis.) in a 0.5 ml microfuge tube. To the DNA admixture, 8 μl of a 2.5 mMsolution of dNTPs (dATP, dCTP, dGTP, dTTP) were admixed to result in afinal concentration of 200 micromolar (μM). Three μl (equivalent to 60picomoles (pM)) of the G-3(F) primer and 3 μl (60 pM) of the 3' backwardG-3(B) primer were admixed into the DNA solution. To the admixture, 73μl of sterile water and 1 μl/5 units of polymerase (Promega Biotech)were added. Two drops of mineral oil were placed on top of the admixtureand 40 rounds of PCR amplification in a thermocycler were performed. Theamplification cycle consisted of 52° C. for 2 minutes, 72° C. for 1.5minutes and 91° C. for 2 minutes. The resultant PCR modified cpIIImembrane anchor domain DNA fragment from M13mp 18 containing sampleswere then purified with Gene Clean (BIO101, La Jolla, Calif.), extractedtwice with phenol/chloroform, once with chloroform followed by ethanolprecipitation and were stored at -70° C. in 10 mM Tris-HCl at pH 7.5,and 1 mM EDTA.

The resultant PCR modified cpIII DNA fragment having Spe I and Nhe Isites in the 5' and 3' ends, respectively, of the fragment was verifiedby electrophoresis in a 1% agarose gel. The area in the agarosecontaining the modified cpIII DNA fragment was isolated from theagarose. The resultant amplified PCR fragment also contained nucleotidesequences for encoding a five amino acid tether composed of four glycineresidues and one serine juxtaposed between the heavy chain and cpIIIencoding domains. Once expressed, the five amino acid residue sequencelacking an orderly secondary structure served to minimize theinteraction between the Fab and cpIII domains.

A second PCR amplification using the primer pairs, Lac-F (SEQUENCE IDNo. 23) and Lac-B (SEQUENCE ID No. 24) listed in Table 3, was performedon a separate aliquot of M13mp 18 replicative form template DNA toamplify the LacZ promoter, operator and Cap-binding site having a 5' NheI site and a 3' EcoR I site. The primers used for this amplificationwere designed to incorporate a Nhe I site on the 5' end of the amplifiedfragment to overlap with a portion of the 3' end of the cpIII genefragment and of the Nhe I site 3' to the amplified cpIII fragment. Thereaction and purification of the PCR product was performed as describedabove.

An alternative Lac-B primer for use in constructing the cpIII membraneanchor and LacZ promotor region was Lac-B' as shown in Table 3. Theamplification reactions were performed as described above with theexception that in the second PCR amplification, Lac-B' was used withLac-F instead of Lac-B. The use of Lac-B' resulted in a LacZ regionlacking 29 nucleotides on the 3' end but was functionally equivalent tothe longer fragment produced with the Lac-F and Lac-B primers.

The products of the first and second PCR amplifications using the primerpairs G-3(F) and G-3(B) and Lac-F and Lac-B were then recombined at thenucleotides corresponding to cpIII membrane anchor overlap and Nhe Irestriction site and subjected to a second round of PCR using the G-3(F)(SEQUENCE ID No. 21) and Lac-B (SEQUENCE ID No. 24) primer pair to forma recombined PCR DNA fragment product consisting of the following: a 5'Spe I restriction site; a cpIII DNA membrane anchor domain beginning atthe nucleotide residue sequence which corresponds to the amino addresidue 198 of the entire mature cpIII protein; an endogenous stop siteprovided by the membrane anchor at amino acid residue number 112; a NheI restriction site, a LacZ promoter, operator and Cap-binding sitesequence; and a 3' EcoR I restriction site.

To construct a phagemid vector for the coordinate expression of a heavychain-cpIII fusion protein with kappa light chain, the recombined PCRmodified cpIII membrane anchor domain DNA fragment was then restrictiondigested with Spe I and EcoR I to produce a DNA fragment for directionalligation into a similarly digested pComb2 phagemid expression vectorhaving only one Spe I site prepared in Example 1a4) to form a pComb2-III(also referred to as pComb2-III) phagemid expression vector. Thus, theresultant ampicillin resistance conferring pComb2-3 vector, having onlyone Spe I restriction site, contained separate LacZ promoter/operatorsequences for directing the separate expression of the heavy chain(Fd)-cpIII fusion product and the light chain protein. The expressedproteins were directed to the periplasmic space by pelB leader sequencesfor functional assembly on the membrane. Inclusion of the phage F1intergenic region in the vector allowed for packaging of single strandedphagemid with the aid of helper phage. The use of helper phagesuperinfection lead to expression of two forms of cpIII. Thus, normalphage morphogenesis was perturbed by competition between the Fab-cpIIIfusion and the native cpIII of the helper phage for incorporation intothe virion for Fab-cpVIII fusions. In addition, also contemplated foruse in this invention are vectors conferring chloramphenicol resistanceand the like.

A more preferred phagemid expression vector for use in this inventionhaving additional restriction enzyme cloning sites, designatedpComb-III' or pComb2-3', was prepared as described above for pComb2-3with the addition of a 51 base pair fragment from pBluescript asdescribed by Short, et al., Nuc. Acids Res., 16:7583-7600 (1988) andcommercially available from Stratagene. To prepare pComb2-3', pComb2-3was first digested with Xho I and Spe I restriction enzymes to form alinearized pComb2-3. The vector pBluescript was digested with the sameenzymes releasing a 51 base pair fragment containing the restrictionenzyme sites Sal I, Acc I, Hinc II, Cla I, Hind II, EcoR V, Pst I, Sma Iand BamH I. The 51 base pair fragment was ligated into the linearizedpComb2-3 vector via the cohesive Xho I and Spe I termini to formpComb2-3'.

                                      TABLE 3                                     __________________________________________________________________________    SEQ ID NO Primer                                                              __________________________________________________________________________    (21).sup.1                                                                          G-3 (F) 5' GAGACGACTAGTGGTGGCGGTGGCTCTCCATTC                                                                    GTTTGTGAATATCAA 3'                    (22).sup.2                                                                          G-3 (B) 5' TTACTAGCTAGCATAATAACGGAATACCCAAAA                                                                    GAACTGG 3'                            (23).sup.3                                                                          LAC-F                                                                               5' TATGCTAGCTAGTAACACGACAGGTTTCCCGAC                                                         TGG 3'                                             (24).sup.4                                                                          LAC-B                                                                               5' ACCGAGCTCGAATTCGTAATCATGGTC 3'                                 (25)5 LAC-B'                                                                             5' AGCTGTTGAATTCGTGAAATTGTTATCCGCT 3'                              __________________________________________________________________________     F Forward Primer                                                              B Backward Primer                                                             1    From 5' to 3': Spe I restriction site sequence is single underlined;     the overlapping sequence with the 5' end of cpIII is double underlined        2    From 5' to 3': Nhe I restriction site sequence is single underlined;     the overlapping sequence with 3' end of cpIII is double underlined.           3    From 5' to 3': overlapping sequence with the 3' end of cpIII is          double underlined; Nhe I restriction sequence begins with the nucleotide      residue "G" at position 4 and extends 5 more residues = GCTAGC.               4    EcoR I restriction site sequence is single underlined.                   5    Alternative backwards primer for amplifying LacZ; EcoR I restriction     site sequence is single underlined.                                      

EXAMPLE 2 ISOLATION OF HUMAN HSV-SPECIFIC MONOCLONAL ANTIBODIES PRODUCEDFROM THE DICISTRONIC EXPRESSION VECTOR,PCOMB2-3

In practicing this invention, the heavy (Fd consisting of V_(H) andC_(H) 1) and light (kappa) chains (V_(L), C_(L)) of antibodies are firsttargeted to the periplasm of E. coli for the assembly of heterodimericFab molecules. In order to obtain expression of antibody Fab librarieson a phage surface, the nucleotide residue sequences encoding either theFd or light chains must be operatively linked to the nucleotide residuesequence encoding a filamentous bacteriophage coat protein membraneanchor. A coat protein for use in this invention in providing a membraneanchor is III (cpIII or cp3). In the Examples described herein, methodsfor operatively linking a nucleotide residue sequence encoding a Fdchain to a cpIII membrane anchor in a fusion protein of this inventionare described.

In a phagemid vector, a first and second cistron consisting oftranslatable DNA sequences are operatively linked to form a dicistronicDNA molecule. Each cistron in the dicistronic DNA molecule is linked toDNA expression control sequences for the coordinate expression of afusion protein, Fd-cpIII, and a kappa light chain.

The first cistron encodes a periplasmic secretion signal (pelB leader)operatively linked to the fusion protein, Fd-cpIII. The second cistronencodes a second pelB leader operatively linked to a kappa light chain.The presence of the pelB leader facilitates the coordinated but separatesecretion of both the fusion protein and light chain from the bacterialcytoplasm into the periplasmic space.

In this process, the phagemid expression vector carries an ampicillinselectable resistance marker gene (beta lactamase or bla) in addition tothe Fd-cpIII fusion and the kappa chain. The f1 phage origin ofreplication facilitates the generation of single stranded phagemid. Theisopropyl thiogalactopyranoside (IPTG) induced expression of adicistronic message encoding the Fd-cpIII fusion (V_(H), C_(H1), cpIII)and the light chain (V_(L), C_(L)) leads to the formation of heavy andlight chains. Each chain is delivered to the periplasmic space by thepelB leader sequence, which is subsequently cleaved. The heavy chain isanchored in the membrane by the cpIII membrane anchor domain while thelight chain is secreted into the periplasm. The heavy chain in thepresence of light chain assembles to form Fab molecules. This sameresult can be achieved if, in the alternative, the light chain isanchored in the membrane via a light chain fusion protein having amembrane anchor and heavy chain is secreted via a pelB leader into theperiplasm.

With subsequent infection of E. coli with a helper phage, as theassembly of the filamentous bacteriophage progresses, the coat proteinIII is incorporated on the tail of the bacteriophage.

a. Preparation of Lymphocyte RNA

Five milliliters of bone marrow was removed by aspiration from a HIV-1seropositive individual exhibiting a high titer (greater than 1:80) ofantibodies to Herpes simplex virus (hereinafter referred to as HSV).Total cellular RNA was prepared from the bone marrow lymphocytes asdescribed above using the RNA preparation methods described byChomczynski, et al., Anal Biochem., 162:156-159 (1987) and using the RNAisolation kit (Stratagene) according to the manufacturer's instructions.Briefly, for immediate homogenization of the cells in the isolated bonemarrow, 10 ml of a denaturing solution containing 3.0 M guanidiniumisothiocyanate containing 71 μl of beta-mercapto- were admixed to theisolated bone marrow. One ml of sodium acetate at a concentration of 2 Mat pH 4.0 was then admixed with the homogenized cells. One ml of phenolthat had been previously saturated with H₂ O was also admixed to thedenaturing solution containing the homogenized spleen. Two ml of achloroform:isoamyl alcohol (24:1 v/v) mixture was added to thishomogenate. The homogenate were mixed vigorously for ten seconds andmaintained on ice for 15 minutes. The homogenate was then transferred toa thick-walled 50 ml polypropylene centrifuged tube (Fisher ScientificCompany, Pittsburgh, Pa.). The solution was centrifuged at 10,000×g for20 minutes at 4° C. The upper RNA-containing aqueous layer wastransferred to a fresh 50 ml polypropylene centrifuge tube and mixedwith an equal volume of isopropyl alcohol. This solution was maintainedat -20° C. for at least one hour to precipitate the RNA. The solutioncontaining the precipitated RNA was centrifuged at 10,000×g for twentyminutes at 4° C. The pelleted total cellular RNA was collected anddissolved in 3 ml of the denaturing solution described above. Three mlof isopropyl alcohol were added to the re-suspended total cellular RNAand vigorously mixed. This solution was maintained at -20° C. for atleast 1 hour to precipitate the RNA. The solution containing theprecipitated RNA was centrifuged at 10,000×g for ten minutes at 4° C.The pelleted RNA was washed once with a solution containing 75% ethanol.The pelleted RNA was dried under vacuum for 15 minutes and thenre-suspended in dimethyl pyrocarbonate-treated (DEPC-H₂ O) H₂ O.

Messenger RNA (mRNA) enriched for sequences containing long poly Atracts was prepared from the total cellular RNA using methods describedin Molecular Cloning: A Laboratory Manual, Maniatis, et al., eds., ColdSpring Harbor, N.Y., (1982). Briefly, one half of the total RNA isolatedfrom a single donor prepared as described above was resuspended in oneml of DEPC-H₂ O and maintained at 65° C for five minutes. One ml of 2×high saft loading buffer consisting of 100 mM Tris-HCl, 1 M NaCl, 2.0 mMEDTA at pH 7.5, and 0.2% SDS was admixed to the resuspended RNA and themixture allowed to cool to room temperature.

The total purified mRNA was then used in PCR amplification reactions asdescribed in Example 2b. Alternatively, the mRNA was further purified topoly A+ RNA by the following procedure. The total MRNA was applied to anoligo-dT (Collaborative Research Type 2 or Type 3) column that waspreviously prepared by washing the oligo-dT with a solution containing0.1 M sodium hydroxide and 5 mM EDTA and then equilibrating the columnwith DEPC-H₂ O. The eluate was collected in a sterile polypropylene tubeand reapplied to the same column after heating the eluate for 5 minutesat 65° C. The oligo-dT column was then washed with 2 ml of high saltloading buffer consisting of 50 mM Tris-HCl at pH 7.5, 500 mM sodiumchloride, 1 mM EDTA at pH 7.5 and 0.1% SDS. The oligo dT column was thenwashed with 2 ml of 1× medium salt buffer consisting of 50 mM Tris-HClat pH 7.5, 100 mM, 1 mM EDTA and 0.1% SDS. The messenger RNA was elutedfrom the oligo-dT column with 1 ml of buffer consisting of 10 mMTris-HCl at pH 7.5, 1 mM EDTA at pH 7.5, and 0.05% SDS. The messengerRNA was purified by extracting this solution with phenol/chloroformfollowed by a single extraction with 100% chloroform. The messenger RNAwas concentrated by ethanol precipitation and resuspended in DEPC H₂ O.

The resultant purified mRNA contained a plurality of anti-herpes simplexvirus (HSV) antibodies encoding V_(H) and V_(L) sequences forpreparation of an anti-HSV Fab DNA library.

EXAMPLE 3 a. Library construction, screening and Fab production

The preparation of human antibody Fab libraries displayed on the surfaceof M13 phage has been described above. The library was constructed as anIgG1×Fab library using bone marrow lymphocyte RNA of a long termasymptomatic HIV-1 positive individual. Antigen binding phage wereselected against HSV-1 and -2 viral lysates (monkey kidney epithelialcells (VERO), 36 hr post infection, were pelleted and lysed usingphosphate-buffered saline containing 1% sodium deoxycholate, 1% NP 40(SIGMA), 0.1 mM DIFP and 2 mg/ml aprotinin) bound to EUSA wells (Sigma)through a panning procedure described in Burton, et al., Proc. Nat'l.Acad. Sci. USA, 88:10134-10137, 1991; Barbas, et al., Methods,2:119-124, 1991. Phage from the final round of panning were converted toa soluble Fab expressing phagemid system and these clones selected forreactivity in ELISA with the antigen against which they were panned.Specific antibody was affinity purified from bacterial supernates over aprotein A/G matrix (Shleicher & Schuell) as described in Williamson, etal., Proc. Nat'l. Acad. Sci. USA, 90:4141-4145, 1993.

b. Viruses and Cells

Vero cells were grown in RPMO 1640 supplemented with 5% fetal calf serum(FCS). HSV-1 and HSV-2 (strain F and G, respectively, ATCC, Rockville,Md.) were infected into Vero cells and virus titers were determined byplaque-assay and expressed as pfu ml-¹

c. Neutralization Activity

Crude E. coli extracts, positive in an ELISA screen using the sameantigen preparation against which the library was panned, were testedfor their ability to neutralize HSV-1 or HSV-2 at 1:5-1:10 dilutionsaccording to the procedure described below. The Fabs that displayedneutralizing activity were affinity purified and their neutralizingtiters were determined as follows. About 250 pfu of HSV-1 or HSV-2 wereincubated with serial dilutions of recombinant Fabs for 1 hr at 37° C.and then adsorbed for 1 hr at 37° C. on Vero cell monolayers grown insix well plates. After adsorption, the inoculum was removed and thecells washed and overlaid with MEM containing 0.5% agarose and 2% FCS.After 72 hrs, the plates were fixed with 10% formaldehyde inphosphate-buffered saline (PBS) for 30 min, the nutrient agar overlaywas removed and the cells were strained with a 1% solution of crystalviolet in 70% methanol for 30 min. The stained monolayers were thenwashed and the plaques were counted.

d. Inhibition of Plaque Development Assay

Monolayers of Vero cells were infected with of 50-100 pfu of HSV-1 for 3hrs at 37° C. They were then washed and the medium replaced withnutrient agar containing 25, 5 or 1 μg/ml of recombinant Fab. After 72hrs or 86 hrs, they were fixed and stained as described above. Plaquediameter was measured with a digital caliper (Mitutoyo, Japan). At least10 plaques were measured per well. Plaques below 0.2 mm in diameter wereconsidered abortive and therefore not counted. Statistical calculationswere performed by analysis of variants (Sheffe F-test).

e. Post-attachement Neutralization Assay

About 250 pfu of HSV-1 were adsorbed at 4° C. for 90 min on Veromonolayers prechilled at 4° C. for 15 min. The inoculum was then removedand the cells washed and overlaid with medium containing serialdilutions of recombinant Fab (5, 1, 0.2, 0.04 μg/ml) at 4° C. to preventpenetration of virus. After 90 min the Fab-contaning medium was removedand after washing, replaced with nutrient agar. For the purpose ofcontrol, equal amounts of virus were preincubated at 4° C. with serialdilutions (5, 1, 0.2, 0.04 μg/ml) of the same Fab (pre-attachmentneutralization). After 90 min these virus/antibody dilutions wereadsorbed onto Vero monolayers (prechilled at 4° C. for 1 hr and 45 min)for 90 min. The inoculum was then removed and the cells washed andoverlaid with nutrient agar. After 72 hr, the monolayers were fixed andstrained as described above for the neutralization assays.

f. Nucleic acid Sequencing

Nucleic acid sequencing was performed with a 373A automated DNAsequencer (Applied Biosystems) using a Taq fluorescent dideoxynucleotideterminator cycle sequencing kit (Applied Biosystems). Primers used forthe elucidation of light and heavy chain sequence have been previouslydescribed in Williamson, et al., (Proc. Nat'l Acad. Sci. USA,90:4141-4145, 1993).

g. Identification of antibody binding protein by immunoprecipitatlon

HSV-2 infected cells were harvested and sonicated in PBS containing 1%sodium deoxycholate, 1% NP40 (Sigma), 0.1 mM di-isopropylfluorophosphate(DIFP) and 2 mg/ml aprotinin. Lysates (50 μl) were then incubated with7μg of recombinant Fab for 1 hour at 4° C. Immune complexes wereprecipitated with an agarose-bound goat anti-human (20 μl) resolved on a10% SDS-PAGE and electro-blotted onto nylon membranes (BioRad) in 1×Towbin buffer. Western blots were performed according to standardprotocols. Briefly, blots were blocked with 5% non-fat dry milk inTris-buffered saline (TBS) and probed with a panel of established mousemonoclonal ant-HSV antibodies (Goodwin Institute) in 1 % non-fat drymilk in TBS containing 0.05% Tween 20. Detection was performed with agoat anti-mouse antibody conjugated to alkaline phosphatase andchemiluminescence (BioRad). Blots were also immunoreacted with a rabbitpolyclonal anti-HSV for the purpose of control and detected with a goatanti-rabbit antibody conjugated to alkaline phosphatase (BioRad).

h. Purification of Fabs

One liter cultures of super broth containing 50 μg/ml carbenicillin and20 mM MgCl₂ were inoculated with appropriate clones and induced 7 hourslater with 2 mM IPTG and grown overnight at 30° C. The cell pellets weresonicated and the supernatant concentrated to 50 ml. The fifteredsupernatants were loaded on a 25 ml protein G-anti-Fab column, washedwith 12 ml buffer at 3 ml/min., and eluted with citric acid, pH 2.3. Theneutralized fractions were then concentrated and exchanged into 50 mMMES pH 6.0 and loaded onto a 2 ml Mono-S column at 1 ml/min. A gradientof 0-500 mM NaCl was run at 1 ml/min with the Fab eluting in the rangeof 200-250 mM NaCl. After concentration, the Fabs were positive whentitered by ELISA against FG and gave a single band at 50 kD by 10-15%SDS-PAGE. Concentration was determined by absorbance measurement at 280nm using an extinction coefficient (1 mg/ml) of 1.35.

EXAMPLE 4 NEUTRALIZING ACTIVITY OF Fab AGAINST HSV

A large panel of human combinatorial antibody Fab fragments specific forHSV-1 and -2 were isolated as described in Example 2. These antibodieswere generated by independently panning an IgG1k Fab library of 2×10⁶members against whole lysate of these two viruses.

Enrichment of antigen specific phage, as determined by the number ofphage eluted from HSV coated ELISA wells, was measured through 4 roundsof library panning. A 25-fold amplification was seen in the case of thepanning with HSV-2 viral lysate, while a 20-fold amplification wasobserved using the HSV-1 viral lysate.

Soluble Fabs were then produced as described in Barbas, et al., Proc.Nat'l Acad. Sci. USA 88:7978-7982, 1991. Briefly, the phage coat proteinIII was excised from the phage display vector and the DNA self-ligatedto give a vector producing soluble Fabs. Subsequently protein synthesiswas induced overnight using IPTG and the bacterial pellet sonicated torelease Fab from the periplasmic space. The Fab supernates were thentested, both in ELISA against the antigen with which they were pannedand in immunofluorescence studies with virus-infected cells. Ten out oftwenty clones taken from the final round of panning with HSV-1 virallysate were positive in both assays, while 15 out of twenty werepositive in the panning against HSV-2 lystate. All clones demonstratingpositive reactivity with one virus type were further shown to becross-reactive with the other in both immunofluorescence and ELISAassays. This probably reflects the known similarity between many of theproteins of HSV-1 and -2.

DNA sequences were determined as described above and the deduced aminoacid sequences of the heavy chain variable domains were determined forseveral of the virus-specific clones (Table 4 and 5). Nine of 18 of theheavy chain sequences obtained from the HSV-2 panning were all quitedifferent from each other. Similarly, 5 of 8 heavy chains taken from theHSV-1 panning were largely unrelated. A comparison of the targets towhich these different antibodies are directed with the serum antibodyreactivity of the donor indicates how accurately the library approachrepresents the humoral response of the donor to virus.

Although virus type cross-reactivity in ELISA was exhibited by all ofthe Fabs described here, only one heavy chain sequence was common toboth pannings. Thus, despite the reported similarity between virions ofHSV-1 and HSV-2 and the observed binding properties of the isolatedFabs, each virus selected distinct antibody molecules from the library.This implies differences between HSV-1 and -2 either in the antigenspresented to the library or in the antibody response to the two viruses.

Neutralizing activity for all positive clones was estimated in plaquereduction and inhibition of plaque development assays of HSV-1 and -2,as described above. Three of the Fabs obtained from the HSV-2 panningexhibited a marked neutralization activity in both assays and with bothvirus types when tested as crude bacterial supernatants in vitro. Theseclones were shown to have identical heavy and light chain sequences.Accordingly, one of these Fab clones (Fab8), was grown in quantity,affinity purified and further characterized.

                                      TABLE 4                                     __________________________________________________________________________    FR1                      CDR1 FR2        CDR2                                 __________________________________________________________________________    ACHSV1 15                                                                           LESGAEVKKPGSSVKVSCK5TSGGAFS                                                                      SYAIN                                                                              WVRQAPGQGLEWHG                                                                           GILPVFGTTNHAQK-                      16, 20, 8                                                                           FQG                                                                     ACHSV1 15                                                                           LEESGAEVKKPGSSVKVSCRASGGTFN                                                                      HYAIS                                                                              WVRQAPGQGLEWHG                                                                           GIFPFRNTAKYAQH-                                                               FQG                                  ACHSV1 13                                                                           LEQSGAEVKKPGSSVKVSCKASGGSFS                                                                      SYAIN                                                                              WVRQAPGQGLEWHG                                                                           GLMPIFGTTNYAQK-                                                               FQD                                  ACHSV1 1                                                                            LEESGGGLVKPGGSLRLSCTASGFIFS                                                                      DFYFS                                                                              WIRQPPGKGLEWLS                                                                           YIGGSHVYTNSADS-                                                               VKG                                  ACHSV1 2                                                                            LESGGGLVQPGGSLRLSCAASGFTFS                                                                       GYAMN                                                                              WVRQAPGKGLEWVS                                                                           AISGNGFSTYYADS-                                                               VKG                                  __________________________________________________________________________           FR3                    CDR3         FR4                                __________________________________________________________________________    ACHSV1 15                                                                            RVTFTADASTSTAYMELSSLRSEDTAVYYCAR                                                                     VGYCSTNGCSLGGMDV                                                                           WGQGTTVIVSS                        16, 20, 8                                                                     ACHSV1 18                                                                            RVTITADESTGTAYMELSSLRSEDTAIYYCAR                                                                     GDTIFGVTHGYYAMDV                                                                           WGQGTTVTVAS                        ACHSV1 13                                                                            RLTITADVSTSTAYMQLTGLTYEDTAMYYCAR                                                                     VAYMLEPTVTAGGLDV                                                                           WGQGTTVTVAS                        ACHSV1 1                                                                             RFTTSRDNAQKSLYLQMNSLRAEDTAVYYCAR                                                                     HSPETDGDTFDY WGQGTLVIVPS                        ACHSV1 2                                                                             RFTISRDNARNTLYLQMNSLRAEDTAVYYCAK                                                                     VLLVATHYYYNGMDV                                                                            WGQGTTVTVSS                        __________________________________________________________________________     Amino acid sequences of heavy chains generated by panning against HSV1        lysate. Clone 13 heavy chain is identical to that of clones 1 and 8           derived from the panning against HSV2.                                   

                                      TABLE 5                                     __________________________________________________________________________    FR1                      CDR1 FR2        CDR2                                 __________________________________________________________________________    ACHSV2 11                                                                           LEESGAEMKKPGSSVRVSCKASGFTFH                                                                      NHAVS                                                                              WVRQAPGEGLEWMG                                                                           GLIPIVGLANLQPRFQG                    ACHSV2                                                                              LEQSGAEVKKPGSSVKVSCKASGGTFS                                                                      SYAIH                                                                              WVRQAPGQGLEWMG                                                                           RITPMFSPAIYAQKFDG                    10, 15, 18,                                                                   20                                                                            ACHSV2 9                                                                            LEESGAEVKKPGESLRITCKAVGYSFT                                                                      NAWIS                                                                              WVRQVPGKGLEWLG                                                                           RINPIDSSRNYSPSFQG                    ACHSV2 8,                                                                           LEQSGAEVKKPGSSVKVSCKASGGSFS                                                                      SYAIN                                                                              WVRQAPGQGLEWMG                                                                           GLMPIFGTTNYAQKFQD                    ACHSV2 7,                                                                           LESGPGLVKTSETLSLTCTVSGGSVSS                                                                      NSDYWA                                                                             WIRQTPGKGLEYFG                                                                           SILFGGTTYYNPSLKS                     16                                                                            ACHSV2 2,                                                                           LESGGGWQPGRSLRLSCAASGFTFR                                                                        TYGMH                                                                              WVRQAPGKGLEWVA                                                                           VISYDGSKNYYADSVKG                    4, 5, 13                                                                      ACHSV2 14                                                                           LEQSGGGWQPGRSLRLSCAASGFTFR                                                                       TYGMH                                                                              WVRQAPGKGLEWVA                                                                           VISYDGSKNYYADSVKG                    ACHSV2 12                                                                           LEQSGAELKRPWSSVKVSCKASGGTLR                                                                      STAVN                                                                              WVRQPPGQGLEWMG                                                                           GLIPLFGTPNYAQKFQG                    ACHSV2 17                                                                           LEQSGAEVKQPGSSMKVSCKVSGGIFR                                                                      TNAFS                                                                              WVRQAPGQGLEWMG                                                                           ISIPMFATVNYAGTFQG                    ACHSV2 6                                                                            LESGPGLVRPSETLFLTCSVSGASIN                                                                       SFFWS                                                                              WIRQSPGKGLEWIG                                                                           HIFHVGITNYNPSLKS                     __________________________________________________________________________              FR3                   CDR3         FR4                              __________________________________________________________________________    ACHSV2 11 RVTISADESTNTAYMEMRSLTSDDTAIYYCVR                                                                    HGDDSSGFPPFDL                                                                              WGQGALVIVSS                      ACHSV2 10, 15, 18,                                                                      RVTITADESTTTAYMEMNSLRSDDTAVYYCAR                                                                    PSAYTGSLAY   WCQGTLVTVSS                      20                                                                            ACHSV2 9  HVTISADTSITSASLHWSSLEASDTAMYYCAR                                                                    HMSDSSGYSNRGAYDI                                                                           WGQGTMVIVPS                      ACHSV2 8, 1                                                                             RLTITADVSTSTAYMQLSGLTYEDTAMYYCAR                                                                    VAYMLEPTVTAGGLDV                                                                           WGQGTTVTVAS                      ACHSV2 7, 16                                                                            RVTMSVDTSTNQFSLDLSSVTMDTAVYYCAR                                                                     HTVTGFLEWSPPNWFFDL                                                                         WGRGTLVTVSS                      ACHSV2 2, 4, 5, 13                                                                      RFTISRDNSKKTLYLQMNSLRAEDTAVYYCAK                                                                    DFWSGSTKNVFDL                                                                              WGQGTLVTVSS                      ACHSV2 14 RFTISRDNSKKTLYLQMNSLRAEDTAVYYCAK                                                                    DFWSGSTKNVFDL                                                                              WGQGTLVTVSS                      ACHSV2 12 RVTFTADESTSTAYMELSSLRSDDTAVYYCAG                                                                    TSRGLNWFDP   WGQGALVTVSS                      ACHSV2 17 RITISADESTSWDMELSSLRPDDTAIYYCAR                                                                     GGRFLEFFEYGLDV                                                                             WGQGTTVIVSS                      ACHSV2 6  RVTMSVDKSKNQFSLRLNSVTMDTAVYYCVR                                                                     VKGLADGGTANWFDP                                                                            WGQGTLVTVSS                      __________________________________________________________________________     Amino acid sequences of heavy chains selected by library panning against      HSV2 lysate.                                                             

The Fab8 antibody was able to recognize both types of the virus. Thisantibody was shown to neutralize HSV-2 (50% inhibition at about 0.05μg/ml) somewhat more efficiently than HSV-1 (50% inhibition at about0.25 μg/ml and 80% inhibition at 0.6 μg/ml) (FIG. 1). FIG. 1 shows theneutralizing activity of Fab8, as measured by plaque reduction. FIG. 1Ashows activity against HSV-1 and FIG. 1B shows activity against HSV-2.Purified Fab8 neutralized HSV-1 with a 50% inhibition at about 0.25μg/ml and with an 80% inhibition at 0.6 μg/ml, while HSV-2 wasneutralized with a 50% inhibition at about 0.05 μg/ml and an 80%inhibition at 0.1 μg/ml.

These figures suggest that Fab8 is approximately an order of magnitudemore potent than most murine neutralizing antibodies described so far(Navarro, et al., Virology, 186:99-112, 1992; Fuller, et al., J. Virol.,55:475-482, 1985), although recently reported anti-gB and anti-gDhumanized murine antibodies may be equally potent (Deschamps, et al.,Proc. Nat'l Acad. Sci. USA, 88:2869-2873, 1991). However, the mouse andhumanized antibodies are bivalent whole IgG molecules rather than humanderived Fab fragments. Also, eukaryotic expression of the recombinantFab of the invention as an intact IgG molecule may significantly enhanceits virus neutralization potency.

The Fab8 antibody also inhibited plaque formation when applied tovirus-infected monolayers (FIG. 2). FIG. 2 shows an inhibition of plaquedevelopment assay. Purified Fab8 inhibited the development of plaqueswhen applied 4 hours post-infection (hpi) on monolayers infected withHSV-1 (FIG. 2A, FIG. 2B) or HSV-2 (FIG. 2C, FIG. 2D) 4 hours postinfection. FIG. 2A shows statistically significant reduction in plaquesize was observed at concentrations of 5 and 1 μg/ml (*=p<0.01), with anapproximate 50% reduction in plaque size at 5 μg/ml. The number ofplaques was also dramatically reduced at Fab concentrations of 5 and 25μg/ml (FIG. 2B, FIG. 2D). At 25 μg/ml and 72 hrs hpi plaque developmentin HSV-2 infected monolayers was completely inhibited (FIG. 2C, FIG.2D). FIG. 2E shows an inhibition of plaque development assay with HSV-2infected monolayers at a number of different Fab concentrations 86 hpi.

At a concentration of 25 μg/ml Fab8 completely abolished HSV-2 plaquedevelopment at 72 hrs post-infection, while a statistically significantreduction in plaque size (>50%) was observed at concentrations of 5μg/ml and 1 μg/ml for both HSV-1 and HSV-2. Since it is accepted thatplaques develop by spreading of virus to adjacent cells, the inhibitionof plaque development assay determines the ability of an antibody toprevent cell-to-cell spread.

Furthermore, this antibody strongly reduced infectivity after HSV-1attachement (FIG. 3). FIG. 3 shows a post-attachment neutralizationassay. Fab8 reduced HSV-1 infectivity after virion attachment. FIG. 3Ashows the percentage of plaque reduction pre- and post-attachment atdifferent Fab concentrations. FIG. 3B shows the post-/pre-attachmentneutralization ratio at different Fab concentrations.

The pre-attachment/post attachment neutralization ratio was over 87% atan antibody concentration of 5 μg/ml, dropping to between 55-60% below 1μg/ml. This suggests that the inhibitory action of the antibody takesplace either at the level of membrane fusion, or during viruspenetration or uncoating.

The protein recognized by Fab8 was identified via immunoprecipitationfrom whole lysate of HSV-2 infected cells. The precipitated proteinswere blotted following resolution through SDS-PAGE and probed with amouse monoclonal ant-gD and a rabbit polyclonal anti-HSV-2 (FIG. 4).FIG. 4 shows the identification of the protein recognized by Fab8.SDS-PAGE of total proteins from HSV-2 infected Vero cells (lanes 1) andof the product of immunoprecipitation with Fab8 (lanes 2). Western blotsperformed in parallel were probed with a mouse monoclonal anti gDantibody (MAB α-gD) and for the purpose of control, a rabbit polyclonalanti-HSV-2 preparation (RAB α-HSV2). The Coomassie stain of a gel run inparallel is also shown. Fab8 immuno-precipitated a band of apparentmolecular weight 48-50 kD which was recognized by a mouse monoclonalspecific for gD, but not by mouse monoclonal antibodies against otherHSV glycoproteins.

The results illustrated in FIG. 4 show that the recombinant Fabrecognizes a protein of mulecular weight approximately 48-50 kD that isalso reactive with murine monoclonal anti-gD. No further proteins weredetected on the blot by the rabbit ant-HSV-2 polyclonal antibodypreparation thus confirming the specificity of the human Fab.

Fab8 has been shown to neutralize virus extremely efficiently and toinhibit viral spread from cell to cell. The demonstration of suchantiviral activity by an Fab offers potential advantages over whole IgGfor some in vivo applications. Although the serum haf life of Fab isdramatically shorter than that of whole IgG, the smaller molecule hasfar greater tissue penetration (Yokota, et al., Cancer Research,52:3401-3408, 1992). The increased penetration of Fab also lends itselfto potential topical applications. In the case of herpes this may takethe form of an antibody cream to treat skin lesions, or as eyedrops forcorneal Infections. Moreover, the use of a Fab may avoid inflammationarising from activation of effector mechanisms.

EXAMPLE 5 KINETICS OF NEUTRALIZATION OF HSV BY Fab8

Neutralization kinetics were performed according to standard techniquesto examine the ability of Fab8 to neutralize HSV. Briefly, 10,000 plaqueforming units (PFU)/ml of HSV type 11 strain G (ATCC VR739), Rockville,Md.) were incubated with three Fab8 different antibody concentrations(2, 6, 18 μg/ml). At different times 100 μl aliquots were removed andimmediately diluted in 10 ml of prechilled PBS at 4° C. to terminate theantibody-antigen reaction. A standard plaque assay method was then usedto determine the residual infectivity in these 1:100 dilutions. Theresidual infectivity expressed as number of plaques/initial viralinoculum (V/Vo) was plotted on a semilogaritmic plot.

FIG. 5A shows the kinetics of neutralization of HSV Type-2, strain G(ATCC, Rockville, Md.) with Fab8. V/Vo is the number of viral plaqueforming units present at time 0 (Vo) or after neutralization (V). FIG.5B shows the kinetics of neutralization of HSV Type-2, strain G (ATCC,Rockville, Md.) with Fab8. The neutralization rate is plotted versus theantibody concentration indicating first order kinetics.

Kinetic neutralization of HSV Type 2 strain G (ATCC, Rockville, Md.)display straight line profiles on semilog plot intercepting theordinates at 1 (FIG. 5A). Furthermore, when the slopes of the linesobtained at different antibody concentrations are plotted versus theantibody concentrations they yield a straight line passing through theorigin (FIG. 5B). The linearity of the two plots is suggestive of singlehit (first order) kinetics, i.e., a single antibody molecule issufficient to produce neutralization (Dulbecco, et al., Virology, 2:162,1956).

TABLE 6 NEUTRALIZATION OF CLINICAL ISOLATES

The recombinant Fab was tested on six low passage clinical isolates ofHSV type I and II which were all efficiently neutralized at similarantibody concentrations.

    ______________________________________                                                                   CLINICAL                                           Patient Initials                                                                          HSV Type       LOCALIZATION                                       ______________________________________                                        F.Z.        II             foreskin                                           R.C.        II             vaginal                                            C.S.        II             vaginal                                            F.S.        I              lip (cold sore)                                    C.B.        I              lip (cold sore)                                    G.D.        I              gingivostomatitis                                  ______________________________________                                    

The ability of this antibody to efficiently neutralize both laboratorystrains and different clinical isolates of HSV type I and II suggeststhat the antibody interacts with a highly conserved and crucialneutralizing epitope and, therefore, may represent a potenttherapeutical tool in clinical practice.

FIG. 6 shows a summary of the results shown in Table 6. The kinetics ofneutralization of HSV Type-2, strain G (ATCC, Rockville, Md.) with Fab8is shown. The plaque reduction is plotted versus antibody concentrationfor 6 different clinical isolates.

Deposit of Materials

The following plasmid has been deposited on Dec. 21, 1993, with theAmerican Type Culture Collection, 1301 Parklawn Drive, Rockville, Md.,20852 USA (ATCC):

    ______________________________________                                        Deposit       ATCC Accession No.                                              ______________________________________                                        Clone FabHSV8 ATCC 69522                                                      ______________________________________                                    

This deposit was made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture for 30 years fromthe date of deposit. The bacteria will be made available by ATCC underthe terms of the Budapest Treaty and applicants assure permanent andunrestricted availability of the progeny of the culture to the publicupon issuance of the pertinent U.S. patent or upon laying open to thepublic of any U.S. or foreign patent application, whichever comes first,and assures availability of the progeny to one determined by the U.S.Commissioner of Patents and Trademarks to be entitled thereto accordingto 35 U.S.C. §122 and the Commissioner's rules pursuant thereto(including 37 CFR §1.14 with particular reference to 886 OG 638). Theassignee of the present application has agreed that if the culturedeposit should die or be lost or destroyed when cultivated undersuitable conditions, it will be promptly replaced on notification with aviable specimen of the same culture. Availability of the depositedstrain is not to be construed as a license to practice the invention incontravention of the rights granted under the authority of anygovernment in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the deposit, since thedeposited embodiment is intended as a single illustration of one aspectof the invention and any plasmid that are functionally equivalent arewithin the scope of this invention. The deposit of material does notconstitute an admission that the written description herein contained isinadequate to enable the practice of any aspect of the invention,including the best mode thereof, nor is it to be construed as limitingthe scope of the claims to the specific illustration that it represents.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and fall within the scope of theappended claims.

SEQUENCE ID LISTING

SEQUENCE ID NO 1 is the amino acid sequence for the CDR3 region of theheavy chain of clone FabHSV 8.

SEQUENCE ID NO 2 is the amino acid sequence for the heavy chain ofFabHSV 8.

SEQUENCE ID NO 3-12 are nucleotide sequences for the polynucleotides forLambda Hc2 construction.

SEQUENCE ID NO 13-20 are nucleotide sequences for the polynucleotidesfor Lambda Lc2 construction.

SEQUENCE ID NO 21-25 are nucleotide sequences for the primers forpCombIII construction.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 25                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 16 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: FabHSV 8-CDR - #3                                        -     (ix) FEATURE:                                                                     (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..16                                                 #ID NO:1: (xi) SEQUENCE DESCRIPTION: SEQ                                      -      Val Ala Tyr Met Leu Glu Pro Thr - # Val Thr Ala Gly Gly Leu Asp        Val                                                                           #   15                                                                        - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 122 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: FabHSV 8                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..122                                                #ID NO:2: (xi) SEQUENCE DESCRIPTION: SEQ                                      -      Leu Glu Gln Ser Gly Ala Glu Val - # Lys Lys Pro Gly Ser Ser Val        Lys                                                                           #   15                                                                        -      Val Ser Cys Lys Ala Ser Gly Gly - # Ser Phe Ser Ser Tyr Ala Ile        Asn                                                                           #                 30                                                          -      Trp Val Arg Gln Ala Pro Gly Gln - # Gly Leu Glu Trp Met Gly Gly        Leu                                                                           #             45                                                              -      Met Pro Ile Phe Gly Thr Thr Asn - # Tyr Ala Gln Lys Phe Gln Asp        Arg                                                                           #         60                                                                  -      Leu Thr Ile Thr Ala Asp Val Ser - # Thr Ser Thr Ala Tyr Met Gln        Leu                                                                           #     80                                                                      -      Ser Gly Leu Thr Tyr Glu Asp Thr - # Ala Met Tyr Tyr Cys Ala Arg        Val                                                                           #   95                                                                        -      Ala Tyr Met Leu Glu Pro Thr Val - # Thr Ala Gly Gly Leu Asp Val        Trp                                                                           #                110                                                          -      Gly Gln Gly Thr Thr Val Thr Val - # Ala Ser                            #            120                                                              - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 32 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: N1                                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..32                                                 #ID NO:3: (xi) SEQUENCE DESCRIPTION: SEQ                                      #          32      TCAA GGAGACAGTC AT                                         - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 36 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: N2                                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..36                                                 #ID NO:4: (xi) SEQUENCE DESCRIPTION: SEQ                                      #       36         CCTA CGGCAGCCGC TGGATT                                     - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 32 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: N3                                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..32                                                 #ID NO:5: (xi) SEQUENCE DESCRIPTION: SEQ                                      #          32      CAAC CAGCCATGGC CC                                         - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 29 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: N6                                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..29                                                 #ID NO:6: (xi) SEQUENCE DESCRIPTION: SEQ                                      #            29    ATGG CTGGTTGGG                                             - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: N7                                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..40                                                 #ID NO:7: (xi) SEQUENCE DESCRIPTION: SEQ                                      #    40            TCCA GCGGCTGCCG TAGGCAATAG                                 - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 38 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: N8                                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..38                                                 #ID NO:8: (xi) SEQUENCE DESCRIPTION: SEQ                                      #     38           GTCT CCTTGAAATA GAATTTGC                                   - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: N9-4                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..40                                                 #ID NO:9: (xi) SEQUENCE DESCRIPTION: SEQ                                      #    40            GATT TCTAGACTAG TTACCCGTAC                                 - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 38 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: N10-5                                                    -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..38                                                 #ID NO:10:(xi) SEQUENCE DESCRIPTION: SEQ                                      #     38           TAAC TAGTCTAGAA ATCTCGAG                                   - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 33 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: N11                                                      -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..33                                                 #ID NO:11:(xi) SEQUENCE DESCRIPTION: SEQ                                      #         33       GTTC TTAATAGAAT TCG                                        - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 28 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: N12                                                      -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..28                                                 #ID NO:12:(xi) SEQUENCE DESCRIPTION: SEQ                                      #             28   AGAA CCGTAGTC                                              - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 34 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..34                                                 #ID NO:13:(xi) SEQUENCE DESCRIPTION: SEQ                                      #        34        CGCC AAGGAGACAG TCAT                                       - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 36 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..36                                                 #ID NO:14:(xi) SEQUENCE DESCRIPTION: SEQ                                      #       36         CCTA CGGCAGCCGC TGGATT                                     - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..31                                                 #ID NO:15:(xi) SEQUENCE DESCRIPTION: SEQ                                      #          31      CAAC CAGCCATGGC C                                          - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 30 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..30                                                 #ID NO:16:(xi) SEQUENCE DESCRIPTION: SEQ                                      #           30     GAGT TAAGCGGCCG                                            - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 48 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..48                                                 #ID NO:17:(xi) SEQUENCE DESCRIPTION: SEQ                                      #                48GTCT CCTTGGCGAC TAGTTTAGAA TTCAAGCT                        - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..40                                                 #ID NO:18:(xi) SEQUENCE DESCRIPTION: SEQ                                      #    40            TCCA GCGGCTGCCG TAGGCAATAG                                 - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..27                                                 #ID NO:19:(xi) SEQUENCE DESCRIPTION: SEQ                                      #             27   GGCT GGTTGGG                                               - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..24                                                 #ID NO:20:(xi) SEQUENCE DESCRIPTION: SEQ                                      #                24TCTA GAAC                                                  - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 48 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..48                                                 #ID NO:21:(xi) SEQUENCE DESCRIPTION: SEQ                                      #                48GCGG TGGCTCTCCA TTCGTTTGTG AATATCAA                        - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..40                                                 #ID NO:22:(xi) SEQUENCE DESCRIPTION: SEQ                                      #    40            TAAC GGAATACCCA AAAGAACTGG                                 - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 36 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..36                                                 #ID NO:23:(xi) SEQUENCE DESCRIPTION: SEQ                                      #       36         ACGA CAGGTTTCCC GACTGG                                     - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..27                                                 #ID NO:24:(xi) SEQUENCE DESCRIPTION: SEQ                                      #             27   TAAT CATGGTC                                               - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..31                                                 #ID NO:25:(xi) SEQUENCE DESCRIPTION: SEQ                                      #          31      AAAT TGTTATCCGC T                                          __________________________________________________________________________

We claim:
 1. A human monoclonal antibody which neutralizes both Herpessimplex virus (HSV) Type 1 and Type 2, binds to an epitope present onglycoprotein D, has the binding specificity of an Fab fragment producedby ATCC 69522, and has heavy chains with a CDR of SEQ ID NO:1.
 2. Thehuman monolonal antibody of claim 1, which is an Fab fragment.
 3. Thehuman monoclonal antibody of claim 2, wherein the monoclonal antibody isan Fab fragment produced by ATCC
 69500. 4. The human monoclonal antibodyof claim 1, wherein the heavy chain comprises a CDR3 polypeptidesequence as in SEQUENCE ID No.
 1. 5. The monoclonal antibody of claim 1wherein the antibody is a single chain antibody.
 6. A peptide whichbinds to an epitope present on glycoprotein D of both antigenic Type 1and Type 2 of Herpes simplex virus (HSV) wherein the peptide has anamino acid sequence of SEQ ID NO:1.
 7. A pharmaceutical compositioncomprising at least one dose of an immunotherapeutically effectiveamount of the monoclonal antibody of claim 1 in a pharmacologicalcarrier.
 8. A kit useful for the detection of Herpes simplex virus (HSV)in a source suspected of containing HSV, the kit comprising carriermeans being compartmentalized to receive in close confinement thereinone or more containers comprising a container containing the monoclonalantibody of claim 1.